Synthetic methods and derivatives of triphosphate oligonucleotides

ABSTRACT

The invention features a oligonucleotide of formula I, or pharmaceutically acceptable salts, or prodrugs thereof: 
     
       
         
         
             
             
         
       
         
         
           
             which are capable of inducing an anti-viral or an antibacterial response, in particular, the induction of type I IFN, IL-18 and/or IL-1β by binding to RIG-I. The invention relates to methods of making and using modified oligonucleotide comprising at least one triphosphate or analogs thereof. The invention further relates to methods for treating various disorders and diseases such as viral infections, bacterial infections, parasitic infections, tumors, allergies, autoimmune diseases, immunodeficiencies and immunosuppression.

PRIORITY CLAIM

This application claims priority to PCT Application No.PCT/US2009/055775, filed Sep. 2, 2009, which claims priority to U.S.Provisional Application No. 61/093,642, filed Sep. 2, 2008, both ofwhich are herein incorporated by reference in their entirety.

TECHNICAL FIELD

The invention relates to methods of making and using modified nucleicacid comprising at least one triphosphate or analogs thereof. Thisinvention also relates to said iRNA agents that are modified so as toeither stimulate or inhibit the immune system of a subject. Thisinvention describes the use of iRNA and RNA agents which are capable ofinducing an anti-viral or an antibacterial response, in particular, theinduction of type I IFN, IL-18 and/or IL-1β by modulating RIG-I. Thisinvention also relates to the use of iRNA agents thatsequence-specifically target the mRNA of certain immune-related anddiseases such as viral infections, bacterial infections, parasiticinfections, tumors, allergies, autoimmune diseases, immunodeficienciesand immunosuppression.

BACKGROUND

Double-stranded RNA molecules (dsRNA) can block gene expression byvirtue of a highly conserved regulatory mechanism known as RNAinterference (RNAi). Briefly, the RNA III Dicer enzyme processes dsRNAinto small interfering RNA (siRNA) of approximately 22 nucleotides. ThesiRNA is assembly into a protein/RNA complex called RNA-inducedsilencing complex (RISC; Bartel, Cell, 2004, 116, 281-297). One of thestrands of the siRNA duplex, the antisense strand or guide strand,hybridizes to a messenger RNA (mRNA) through formation of the specificWatson-Crick base pairs. The endonuclease component of the RISC, Slicer,cleaves the targeted mRNA (Meister et al., Mol. Cell, 2004, 15,185-197.). The antisense strand of the siRNA duplex is not cleaved orotherwise degraded in this process, and the RISC that includes thisantisense strand can subsequently cleave additional complementary mRNAs.

Many diseases (e.g., cancers, hematopoietic disorders, endocrinedisorders, and immune disorders) arise from the abnormal or otherwiseunwanted expression or activity of a particular gene or group of genes.For example, disease can result through misregulated gene expression,expression of a mutant form of a protein, or expression of viral,bacterial or other pathogen-derived genes. The RNAi pathway can be usedto inhibit or decrease the unwanted expression of such genes (Agrawal etal., Microbiol Mol Biol Rev., 2003, 67, 657-685; Alisky & Davidson, Am.J. Pharmacogenomics, 2004, 4, 45-51).

A number of receptor proteins have evolved that take part in nucleicacid recognition. Recent studies indicate that one of the most importantprotein receptors for antiviral defense is the retinoic-acid-inducibleprotein I (RIG-I), a member of the helicase family containing twocaspase-recruitment domains (CARDs) and a DExD/H-box helicase domain (M.Yoneyama et al., Nat Immunol 5, 730 (JuI, 2004)). RIG-1-mediatedrecognition of a specific set of RNA viruses (flaviviridae,paramyxoviridae, orthomyxoviridae and rhabdoviridae) (M. Yoneyama etal., Nat Immunol 5, 730 (JuI, 2004); R. Sumpter, Jr. et al., J Virol 79,2689 (March, 2005); H. Kato et al., Nature 441, 101 (Apr. 9, 2006)) hasa critical role in antiviral host defense in vitro and in vivo. A secondmember of the helicase family, MDA-5, is responsible for the antiviraldefense against a reciprocal set of RNA viruses (picomaviridae)(H. Katoet al., Nature 441 (7089): 101-105, Apr. 9, 2006).

In addition to RIG-I and MDA-5, the four members of the Toll-likereceptor (TLR) family, TLR3, TLR7, TLR9 and TLR9, are also known to beinvolved in viral nucleic acid recognition. RIG-I and MDA-5 differ fromthe TLRs in their subcellular localization, expression patem, signaltransduction pathways and ligands.

While RIG-I and MDA-5 are cytosolic receptors, TLR3, TLR7, TLR8 and TLR9are located in the endosomal membrane.

While TLRs are mainly expressed on certain defined immune cell subsets(i.e. TLR9 restricted to PDC and B cells), RIG-I and MDA-5 are expressedin both immune and nonimmune cells (H. Kato et al., Immunity 23, 19(JuI₁ 2005)).

Besides distinct expression profiles and cellular localization,signalling of endosomal TLRs and the two cytoplasmic receptors RIG-I andMDA-5 differs. While TLR3 signals via TRIF and TLR7, TLR8 and TLR9signal via MyD88, RIG-I recruits a CARD-containing adaptor.

The iRNA agents described in this patent are duplexes of chemicallysynthesized oligoribonucleotides. One or both strands of the duplex maybe chemically modified to alter the properties of the duplex asdescribed below. In addition to RISC-mediated cleavage of mRNArecognized in a sequence specific manner, the duplexes may have aneffect on the immune system. This invention relates to iRNA agents thatare modified so as to either stimulate or inhibit the immune system of asubject. These iRNA agents may have a dual function and alsosequence-specifically target an mRNA of a gene not related to the immunecascade. These immune stimulatory or inhibitory iRNA agents may also beused in conjunction with a second therapeutic, which may be achemotherapeutic agent, an antibiotic, or a second iRNA agent. Thisinvention also relates to the use of iRNA agents that sequencespecifically target the mRNAs of certain immune-related proteins, forexample TLR 3, TLR 7, TLR 8 and TLR 9, for degradation via a RISC-basedmechanism.

Pathogen recognition is mediated by the innate recognition arm of theimmune system. Unlike the adaptive immune system, innate immunity doesnot have the potential for recognition of all possible antigens.Instead, a few highly conserved structures present in many differentmicroorganisms are recognized. The innate immune response is based onthe recognition of ligands by pathogen recognition receptors (PRRs) onepithelial and immune cells. The ligands include lipopolysaccharide(LPS) from the gram-negative cell wall, peptidoglycan, lipotechoic acidsfrom the gram-positive cell wall, mannose sugar, bacterial DNA,N-formylmethionine, double-stranded RNA from viruses, and glucans fromfungal cell walls. Innate immunity is activated immediately or withinseveral hours after exposure to a recognized ligand.

Cells involved in the innate immune response include phagocytic cells,basophils, mast cells, eosinophils, and natural killer cells (NK cells).There are two functionally different classes of cell surfacepattern-recognition receptors on these cells: endocyticpattern-recognition receptors and signaling pattern-recognitionreceptors. For the purpose of this document, signalingpattern-recognition receptors, in general, and the toll-like receptors,in particular, are most important. The toll-like receptors (TLRs) bindseveral different molecules of microbial origin including DNA frombacteria and double-stranded RNA (Janssens & Beyaert, Clin. Microbiol.Rev., 2003, 16, 637-646).

When a ligand binds to a TLR, a signal is transmitted to the nucleus andgenes coding for the synthesis of intracellular regulatory molecules areexpressed (Ulevitch, Nat. Rev. Immunol., 2004, 4, 512-520). TLRsignaling relies on adaptor proteins including MyD88, Tollip, Mal, andothers. These adaptor proteins activate cellular responses to induceproduction of inflammatory cytokine production, induce maturation ofdendritic cells, and induce production of interferons. The cytokines, inturn, trigger innate immune defenses such as inflammation, fever, andphagocytosis and provide an immediate response against the invadingmicroorganism. TLRs also participate in adoptive immunity by triggeringvarious secondary signals needed for humoral immunity (the production ofantibodies) and cell-mediated immunity (the production of cytotoxicT-lymphocytes and additional cytokines).

The iRNA agents described in this document may activate the innateimmune system through one of the receptors known to bind nucleic acidsor nucleotide analogs. Toll-like receptor 3 (TLR3) is the receptor fordouble-stranded RNA. TLR3 is expressed on dendritic cells, fibroblasts,macrophages, and epithelial cells (Matsumoto et al., Microbial.Immunol., 2004, 48, 147-154). The adaptor molecule for TLR3 is TICAM-1.Binding of TLR3 to TICAM-1 induces multiple signaling cascades thatultimately lead to production of type I interferons (IFN-αβ, Matsumotoet al., Microbial. Immunol., 2004, 48, 147-154). The interferons arecytokines that induce uninfected cells to produce enzymes capable ofdegrading RNA thus preventing viral replication. Interferons alsoactivate a variety of cells important to defense including cytoxicT-lymphocytes, macrophages, and NK cells.

Single-stranded RNA recognition is mediated in mouse by Toll-likereceptor 7 and in humans by TLR-8. In mice, TLR7 binds to the adaptorMyD88 and leads to activation of IFN-α. Diebold et al. (Science, 2004,303, 1529-1531) showed that influenza virus RNA, polyuridylic acid, andin vitro synthesized mRNA all induced production of IFN-α inplasmacytoid dendritic cells. Heil et al. (Science, 2004, 303,1526-1529) showed that guanine- and uridine-rich RNA oligonucleotides of20 residues with phosphorothioate termini stimulated dendritic cells andmacrophages to secrete INF-α and proinflammatory and regulatorycytokines Using TLR-deficient mice, they showed that mouse TLR-7 andhuman TLR-8 were responsible for binding to single-stranded RNA. HumanTLR-7 is also activated by guanine nucleotide analogs (Lee et al., Proc.Natl. Acad. Sci. USA, 2003, 100, 6646-6651).

DNA from bacteria has stimulatory effects on mammalian immune cells.This response depends on the presence of unmethylated CpG dinucleotidesin the bacterial DNA; mammalian DNA has a low frequency of CpGdinucleotides and these are mostly methylated, therefore, mammalian DNAdoes not have immunostimulatory activity. The cellular response to CpGDNA is mediated by TLR9 (Hemmi et al., Nature, 2000, 408, 740-745).Short DNA oligonucleotides with a CpG motif have immune stimulatoryeffects that depend on the bases flanking the CpG dinucleotide, on thenumber and spacing of the CpG motifs, on the presence of poly Gsequences in the ODN, and on the ODN backbone (Krieg et al., Nature,1995, 374, 546-549; Krieg, In: Antisense Drug Technology: Principles,Strategies, and Applications, 2001, ed. Crooke, S. T., pp. 471-516,Marcel Dekker, Inc., New York).

Double-stranded RNA may also activate the innate immune system throughinteraction with a ubiquitously-expressed serine/threonine proteinkinase called PKR. PKR is part of the TLR4 cascade activated by TLR4binding of bacterial LPS. PKR is induced by interferon and activated bydsRNA, cytokines, growth factors, and stress signals. PKR isautophosphorylated and activated upon binding to dsRNA. Activationresults in inhibition of protein synthesis via the phosphorylation ofeIF2a and also induces transcription of inflammatory genes byPKR-dependent signaling of the activation of different transcriptionfactors (Williams, Oncogene, 1999, 18, 6112-6420). PKR up-regulatesNF-κB expression through phosphorylation of its inhibitor IkB (Kumar etal., Proc. Natl. Acad. Sci. USA, 1994, 91, 6288-6292). As few as 11 basepairs of dsRNA can bind to PKR and induce activity, but maximalactivation requires at least 30 base pairs (Manche et al., Mol. Cell.Biol., 1992, 12, 5238-5248; Nanduri et al., EMBO J., 1998, 17,5458-5465). Interestingly, Kim et al. (Nat. Biotechnol., 2004, 22,321-325) recently showed that the 5′ triphosphate on siRNAs transcribedusing T7 RNA polymerase was responsible for type I IFN induction.

There have been reports that siRNA duplexes are able to trigger animmune response in human cells under certain conditions. Sledz et at(Nat. Cell Biol., 2003, 5, 834-839) reported induction of the interferonsystem with each of the six different siRNA duplexes tested. Bridge etal. (Nat. Genet., 2003, 34, 263-264) reported that some, but not all, ofthe hairpin siRNAs delivered using viral vectors induced expression ofan interferon-stimulated gene. The concentration of the siRNA duplex isdirectly correlated with the extent of the immune response. These tworeports showed that the siRNA duplexes activated PKR. In contrast, datafrom Kariko et al. (Cells Tissues Organs, 2004, 177, 132-138) implicatedthe TLR3 pathway in siRNA induction of innate immunity. Kariko et al.(Cells Tissues Organs, 2004, 177, 132-138) showed that TLR3 activationwas concentration dependent, but the requirements for optimal TLR3activation by dsRNA are presently unknown.

Activation of the innate immune response is advantageous in diseasesranging from viral infections to cancer. Activation of innate immunityby dsRNA and CpG DNA promoted antitumor effects in a mouse model(Whitmore et al., Cancer Res., 2004, 64, 5850-5860). iRNA agents shouldbe potent adjuvants for vaccination against a variety of bacterial andviral pathogens. Such adjuvants should activate innate immunity, which,in turn should shape the adaptive immune response. Some iRNA sequencesand modifications may better activate the innate immune response thanothers. This activation could come via any of the Toll-like receptorpathways that are known to bind nucleic acids: TLR3, the receptor fordouble-stranded RNA; TLR8, the receptor for single-stranded RNA; TLR9,the receptor with a preference for unmethylated CpG DNA; or PKR, theprotein kinase activated by dsRNA. Each of these proteins is known tobind to molecules similar to the iRNA agents described.

There are a number of reasons that suppression of the innate immunesystem might be advantageous. As stimulation of the innate immune systemresults in inflammation, depression of this system when it isnon-productive may benefit patients with asthma, serious local orsystemic infections, or chronic inflammatory diseases such asinflammatory bowel disease, chronic obstructive pulmonary disease, andarthritis. Compounds that suppress the immune system may also be usefulin treatment of cancer. In particular, disruption of the PKR pathway,which leads to activation of NF-κB, is attractive. NF-κB leads toinhibition of apoptosis that would otherwise eliminate defective cells(Pikarsky et al., Nature, 2004, 431, 461-466) and also promotesmetastatic growth (Huber et al., J. Clin. Invest., 2004, 114, 569-581).

SUMMARY

The present invention provides a novel class of iRNA agents that arecapable of inducing an anti-viral or an antibacterial response, inparticular, the induction of type I IFN, IL-18 and/or IL-1β bymodulating RIG-I.

In one embodiment, the iRNA agents of the present invention arerepresented by formula I, as illustrated below:

or pharmaceutically acceptable salts or prodrugs thereof, whereinQ₂ and Q₃ are each, independently NH, O or S;X and Y are each, independently, OH, O⁻, OR¹, O⁻, SH, S⁻, Se, BH₃, BH₃⁻, H, N(R²)₂, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each ofwhich may be optionally substituted, where R¹ is independently alkyl,cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, and R² is independently hydrogen, alkyl,cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, or OH;Q₄ and Q₅ are each independently O, CH₂, CHMe, CMe₂, CHF, CF₂, NH, NR¹,or S;

Q₁ is OH, O⁻, OR¹, S⁻, SH, SR¹;

n is 0, 1, 2, 3, 4 or 5; wherein each repeating unit can be the same ordifferent;A is absent or selected from the group consisting of single strandedoligonucleotide and double stranded oligonucleotide, each of which maybe chemically modified;B is absent or a linker/spacer;E is a single stranded oligonucleotide or a double strandedoligonucleotide, each of which may be chemically modified or conjugatedwith a ligand;with a proviso that when A and B are both absent and n is 0, 1 or 3,then Q₁, Q₂, Q₃, Q₄, Q₅, X and Y cannot be all oxygen.

In another embodiment of the present invention there are disclosedpharmaceutical compositions comprising a therapeutically effectiveamount of a iRNA agent of the invention in combination with apharmaceutically acceptable carrier or excipient. In yet anotherembodiment of the invention are methods of treating diseases such asviral infections, bacterial infections, parasitic infections, tumors,allergies, autoimmune diseases, immunodeficiencies andimmunosuppression. with said pharmaceutical compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1 is an ion-exchange HPLC analysis of purified RNA-Triphosphate

FIG. 2 is an LC-MS analysis of RNA 5′-Triphosphate

DETAILED DESCRIPTION

In a first embodiment of the compounds of the present invention arecompounds represented by formula I as illustrated above, or apharmaceutically acceptable salt, ester or prodrug thereof.

In one embodiment of the iRNA agents of the present invention are agentsrepresented by formula II as illustrated below, or a pharmaceuticallyacceptable salt or prodrug thereof:

Q₂, Q₃ and Q₃₀ are each, independently NH, O or S;

X and Y and Y₁₀ are each, independently, OH, O⁻, OR¹, O⁻, SH, S⁻, Se,BH₃, BH₃ ⁻, H, N(R²)₂, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl,each of which may be optionally substituted, where R¹ is independentlyalkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, and R² is independently hydrogen, alkyl,cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, or OH;

Q₄ and Q₅ are each independently O, CH₂, CHMe, CMe₂, CHF, CF₂, NH, NR₁or S;

Q₁ is OH, O⁻, OR₁, S⁻, SH, SR¹;

W is H, OH or -G-L; where G is selected from the group consisting of—CONH—, —NHCO—, —S—S—, —OC(O)NH—, —NHC(O)O—, —NHC(O)NH—, acetal, ketal,—O—N═C—, —NH—N═C—, —S—, —O—, pyrrolidine, morpholine, piperazine andthiazolidine; and where L is a ligand;provided that when W is OH, then Q₁, Q₂, Q₃, Q₄, Q₅, Q₃₀, X, Y and Y₁₀cannot all be oxygen.

In one embodiment of the iRNA agents of the present invention are agentsrepresented by formula III as illustrated below, or a pharmaceuticallyacceptable salt or prodrug thereof:

Q₂ and Q₃ are each, independently NH, O or S;X and Y and Y₁₀ are each, independently, OH, O⁻, OR₁, O⁻, SH, S⁻, Se,BH₃, BH₃ ⁻, H, N(R²)₂, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl,each of which may be optionally substituted, where R¹ is independentlyalkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, and R² is independently hydrogen, alkyl,cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, or OH;

Q₄ and Q₅ are each independently O, CH₂, CHMe, CMe₂, CHF, CF₂, NH, NR₁or S;

Q₁ is OH, O⁻, OR₁, S⁻, SH, SR¹;

W is each independently H, OH or -G-L; where G is selected from thegroup consisting of —CONH—, —NHCO—, —S—S—, —OC(O)NH—, —NHC(O)O—,—NHC(O)NH—, acetal, ketal, —O—N═C—, —NH—N═C—, —S—, —O—, pyrrolidine,morpholine, piperazine and thiazolidine; and where L is a ligand.

In one embodiment of the iRNA agents of the present invention are agentsrepresented by formula IV as illustrated below, or a pharmaceuticallyacceptable salt or prodrug thereof:

Q₂, Q₃ and Q₃₀ are each, independently NH, O or S;X and Y and Y₁₀ are each, independently, OH, O⁻, OR₁, O⁻, SH, S⁻, Se,BH₃, BH₃ ⁻, H, N(R²)₂, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl,each of which may be optionally substituted, where R¹ is independentlyalkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, and R² is independently hydrogen, alkyl,cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, or OH;Q₄ and Q₅ are each independently O, CH₂, CHMe, CMe₂, CHF, CF₂, NR¹, orS;

Q₁ is OH, O⁻, OR₁, S⁻, SH, SR¹;

R is H, phosphate or phosphorothioate;W and W₁ are each independently H, OH, phosphate, phosphorothioate or-G-L; where G is selected from the group consisting of —CONH—, —NHCO—,—S—S—, —OC(O)NH—, —NHC(O)O—, —NHC(O)NH—, acetal, ketal, —O—N═C—,—NH—N═C—, —S—, —O—, pyrrolidine, morpholine, piperazine andthiazolidine; and where L is a ligand;provided that when W is OH, then Q₁, Q₂, Q₃, Q₄, Q₅, Q₃₀, X, Y and Y₁₀cannot be all oxygen.

In one embodiment of the iRNA agents of the present invention are agentsrepresented by formula IVa as illustrated below, or a pharmaceuticallyacceptable salt or prodrug thereof:

Q₂, Q₃ and Q₃₀ are each, independently NH, O or S;X and Y and Y₁₀ are each, independently, OH, O⁻, OR¹, O⁻, SH, S⁻, Se,BH₃, BH₃ ⁻, H, N(R²)₂, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl,each of which may be optionally substituted, where R¹ is independentlyalkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, and R² is independently hydrogen, alkyl,cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, or OH;Q₄ and Q₅ are each independently O, CH₂, CHMe, CMe₂, CHF, CF₂, NR¹, orS;

Q₁ is OH, O⁻, OR¹, S⁻, SH, SR¹;

W and W₁ are each independently H, OH, phosphate, phosphorothioate or-G-L; where G is selected from the group consisting of —CONH—, —NHCO—,—S—S—, —OC(O)NH—, —NHC(O)—, —NHC(O)NH—, acetal, ketal, —O—N═C—,—NH—N═C—, —S—, —O—, pyrrolidine, morpholine, piperazine andthiazolidine; and where L is a ligand;provided that when W is OH, then Q₁, Q₂, Q₃, Q₄, Q₅, Q₃₀, X, Y and Y₁₀cannot be all oxygen.

In one embodiment of the iRNA agents of the present invention are agentsrepresented by formula V as illustrated below, or a pharmaceuticallyacceptable salt or prodrug thereof:

Q₂ and Q₃ are each, independently NH, O or S;X and Y and Y₁₀ are each, independently, OH, O⁻, OR¹, O⁻, SH, S⁻, Se,BH₃, BH₃ ⁻, H, N(R²)₂, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl,each of which may be optionally substituted, where R¹ is independentlyalkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, and R² is independently hydrogen, alkyl,cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, or OH;Q₄ and Q₅ are each independently O, CH₂, CHMe, CMe₂, CHF, CF₂, NR¹, orS;

Q₁ is OH, O⁻, OR¹, S⁻, SH, SR¹;

R is H, phosphate or phosphorothioate;W and W₁ are each independently H, OH, phosphate, phosphorothioate or-G-L; where G is selected from the group consisting of —CONH—, —NHCO—,—S—S—, —OC(O)NH—, —NHC(O)O—, —NHC(O)NH—, acetal, ketal, —O—N═C—,—NH—N═C—, —S—, —O—, pyrrolidine, morpholine, piperazine andthiazolidine; and where L is a ligand.

In one embodiment of the iRNA agents of the present invention are agentsrepresented by formula VI as illustrated below, or a pharmaceuticallyacceptable salt or prodrug thereof:

Q₂, Q₃ and Q₃₀ are each, independently NH, O or S;X and Y and Y₁₀ are each, independently, OH, O⁻, OR₁, O⁻, SH, S⁻, Se,BH₃, BH₃ ⁻, H, N(R²)₂, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl,each of which may be optionally substituted, where R¹ is independentlyalkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, and R² is independently hydrogen, alkyl,cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, or OH;Q₄ and Q₅ are each independently O, CH₂, CHMe, CMe₂, CHF, CF₂, NR¹, orS;

Q₁ is OH, O⁻, OR₁, S⁻, SH, SR¹;

W, W₁ and W₂ are each independently H, OH, phosphate, phosphorothioateor -G-L; where G is selected from the group consisting of —CONH—,—NHCO—, —S—S—, —OC(O)NH—, —NHC(O)O—, —NHC(O)NH—, acetal, ketal, —O—N═C—,—NH—N═C—, —S—, —O—, pyrrolidine, morpholine, piperazine andthiazolidine; and where L is a ligand;Linker/spacer is selected from the group consisting of phosphate,phosphorothioate, phosphorodithioate, alkylphosphonate, amide, ester,disulfide, thioether, oxime and hydrazone, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclyl and heteroaryl.

In one embodiment of the iRNA agents of the present invention are agentsrepresented by formula VII as illustrated below, or a pharmaceuticallyacceptable salt or prodrug thereof:

Q₂ and Q₃ are each, independently NH, O or S;X and Y and Y₁₀ are each, independently, OH, O⁻, OR₁, O⁻, SH, S⁻, Se,BH₃, BH₃ ⁻, H, N(R²)₂, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl,each of which may be optionally substituted, where R¹ is independentlyalkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, and R² is independently hydrogen, alkyl,cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted, or OH;Q₄ and Q₅ are each independently O, CH₂, CHF, CF₂, NR¹, or S;

Q₁ is OH, O⁻, OR₁, S⁻, SH, SR¹;

W, W₁ and W₂ are each independently H, OH, phosphate, phosphorothioateor -G-L; where G is selected from the group consisting of —CONH—,—NHCO—, —S—S—, —OC(O)NH—, —NHC(O)O—, —NHC(O)NH—, acetal, ketal, —O—N═C—,—NH—N═C—, —S—, —O—, pyrrolidine, morpholine, piperazine andthiazolidine; and where L is a ligand;

Linker/spacer is selected from the group consisting of is selected fromthe group consisting of phosphate, phosphorothioate, phosphorodithioate,alkylphosphonate, amide, ester, disulfide, thioether, oxime andhydrazone, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl andheteroaryl.

Further representative sub species of the present invention are iRNAagents (1)-(15) of the formula (A), where Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, X, Yand Z are delineated in Table 1:

TABLE 1 (A)

X Y Z Q1 Q2 Q3 Q4 Q5 Q6 1 O O S O O O O O O 2 O S S O O O O O O 3 S O SO O O O O O 4 S S O O O O O O O 5 S S S O O O O O O 6 O O S O O O S O O7 O O S S O O O O O 8 S O S S O O O O O 9 S O S S O O S O O 10 O O S S OO S O O 11 O O O S O O O O O 12 O O O S S O O O O 13 O O O S O S O O O14 S O O S O O S O O 15 O O O S O O S O O

Further representative sub species of the present invention are iRNAagents (16)-(31) of the formula (B), where Q₁, Q₂, Q₃, Q₅, Q₆, X, Y andZ are delineated in Table 2:

TABLE 2 (B)

X Y Z Q1 Q2 Q3 Q5 Q6 16 O O O O O O O O 17 O O S O O O O O 18 O S S O OO O O 19 S O S O O O O O 20 S S O O O O O O 21 S S S O O O O O 22 O O SO O O O O 23 O O S S O O O O 24 S O S S O O O O 25 S O S S O O O O 26 OO S S O O O O 27 O O O S O O O O 28 O O O S S O O O 29 O O O S O S O O30 S O O S O O O O 31 O O O S O O O O

In one embodiment, oligonucleotide of the invention can be prepared by aA process of preparing nucleic acid molecule comprising atriphosphosphate or a triphosphate analog comprising the steps of: (a)protecting the 2′ hydroxyl moiety with fluoride labile group or fluoridenon-labile group; (b) converting the 5′ hydroxyl moiety to triphosphateor triphosphate analog with a reagent selected from the group consistingof:

-   -   wherein:    -   R₁₀₀ is independently electron withdrawing group (EWG);    -   R₂₀₀ and R₃₀₀ are each independently haloalkyl, aryl,        substituted aryl, heteroaryl, substituted heteroaryl,        cycloalkyl, substituted cycloalkyl, heterocycle, substituted        heterocyclo;    -   Z₁₀ is O, S, Se, BH₃ or NR;    -   X₄₀ is Cl, dialkylamine or cyclic amine;    -   X₁₀ is Cl, O-aryl or O-substituted aryl;    -   Y₁₀ and Y₂₀ are independently O-substituted alkyl; dialkylamine        or cyclic amine, wherein the nitrogen is connected to the        phosphorus;    -   X₂₀ and X₃₀ is independently O, CH₂, S, NR′, wherein R′ is H or        aliphatic;    -   n is 1, 2, 3, 4, or 5; and    -   s is 0, 1, 2 or 3;        (c) synthesizing said nucleic acid molecule using a method        selected from the group consisting of solid phase        phosphoramidite, solution phase phosphoramidite, solid phase        H-phosphonate, solution phase H-phosphonate, hybrid phase        phosphoramidite, and hybrid phase H-phosphonate-based synthetic        methods; and (d) removing the protecting group(s) and/or solid        support. In one embodiment, the steps of the synthesis can be        interchanged. For example, step (c) can precede step (a) and/or        (b).

Suitable electron withdrawing groups include halogens, NO₂, CN, acyl,and sulfonyl. Suitable dialkylamines include N(i-Pr)₂.

In one embodiment, the sequence and motif of the oligonucleotide can bevary to induce RIG-1 activation in combination with 1) phosphorothioatelinkage (5′-triphosphate with PS linkage—racemic at 5′ and at internalpositions; or 5′-triphosphate with PS linkage—optically pure (R and/or Sconfiguration) at 5′ and at internal positions); 2) chimericoligonucleotides (5′-triphosphate-RNA-DNA-RNA-3′ with CPG motif on theshort DNA part to induce TLR activation. The DNA portion is 4-6nucleotides in length, which exerts minimal effect on RNase H;5′-Triphosphate-RNA-DNA-RNA-3′ without CPG motif on the DNA part thathas the potential to induce RNase H; 5′-triphosphate-RNA-DNA-RNA-3′ withCPG motif on the DNA part that has the potential to induce RNase H andTLR activation; 5′-triphosphate-RNA-DNA-3′ with CPG motif on the shortDNA part to induce TLR activation where the DNA portion is 4-6nucleotides in length, which exerts minimal effect on RNase H;5′-triphosphate-RNA-DNA-3′ without CPG motif on the DNA part that hasthe potential to induce RNase H; 5′-triphosphate-RNA-DNA-3′ with CPGmotif on the DNA part that has the potential to induce RNase H and TLRactivation; 3) 5′-triphosphate oligoribonucleotide—insert sequencemotifs known for immune stimulation; 4) place stimulatingmotif(s)—single motif or combinations there of described above—in thesense strand and preserve antisense strand for RNAi; and 5) placestimulating motif(s)—single motif or combinations there of describedabove—in the sense strand and introduce minimal chemical modification,that retain RNAi activity and that protect antisense strand fromnucleases.

In one embodiment, the preferred oligonucleotide can have all natural2′-deoxyribo and 2′-ribonuclesides, 2′-O-methyl (2′-OMe),2′-O-methoxyethyl(2′-MOE), 2′-deoxy-2′-ribofluoro(2′-F),2′-deoxy-2′-arabinofluoro(2′-araF) sugar modifications and combinationsthere of, with and without phosphorothioate backbone at theinternucleoside linkages.

In one embodiment, the preferred nucleobase modifications includes2-ThioU, 2′-amino-A, pseudouridine, inosine, 5-Me-U, 5-Me-C, chemicallymodified U analogues.

In one embodiment, the preferred Ligands includes PK modulators such aslipophiles, Cholesterol and analogs, bile acids, steroids, circulationenhancers—PEG with different mol. wt. starting from 400 to up to 60,000amu, small molecule protein binders (for e.g, naproxen or ibuprofen) andtargeting ligands for receptor targeting, for e.g., folic acid, GalNAcand mannose.

Evaluation of the iRNA agent can include incubating the modified strand(with or without its complement, but preferably annealed to itscomplement) with a biological system, e.g., a sample (e.g, a cellculture). The biological sample can be capable of expressing a componentof the immune system. This allows identification of an iRNA agent thathas an effect on the component. In one embodiment, the step ofevaluating whether the iRNA agent modulates, e.g, stimulates orinhibits, an immune response includes evaluating expression of one ormore growth factors, such as a cytokine or interleukin, or cell surfacereceptor protein, in a cell free, cell-based, or animal assay. Exemplaryassay methods include ELISA and Western blot analysis. Growth factorsthat could be evaluated include TNFα, IL1α and β, IL2, IL3, IL4, IL5,IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IFNα and β, and IFNγ. Inpreferred embodiments, a test includes evaluating expression of one ormore of the interleukins IL-18, IL-1β, IL-10, IL-12, and IL-6. Relevantcell surface receptors include the toll-like receptors, e.g., TLR1,TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In other preferredembodiments, a test includes evaluating expression of one or more of thetoll-like receptors TL-3, TLR7, TLR8, or TLR9. Ligand interaction withTLR9 stimulates expression of NFκB. Therefore, testing whether an iRNAagent stimulates the immune response can include assaying for NFκBprotein or mRNA expression.

In one embodiment, the step of testing whether the modified iRNA agentmodulates, e.g., stimulates, an immune response includes assaying for aninteraction between the iRNA agent and a protein component of the immunesystem, e.g., a growth factor, such as a cytokine or interleukin, or acell surface receptor protein. For example, the test can includeassaying for an interaction between the modified iRNA and a toll-likereceptor, e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9.In one preferred embodiment, testing includes assaying for aninteraction with a toll-like receptor, e.g., TLR-9. Exemplary assaymethods include coimmunoprecipitation assays, bead-based co-isolationmethods, nucleic acid footprint assays and colocalization experimentssuch as those facilitated by immunocytochemistry techniques.

In one embodiment, the candidate iRNA agent has the ability tosequence-specifically inhibit expression of a particular gene throughthe RNA interference pathway. For example, the iRNA agent can target asequence that encodes a protein component of the immune system, such asa cytokine, cytokine receptor or Toll-like receptor (e.g., TLR 3, TLR 7,TLR 8 and TLR 9,). The ability of an iRNA agent composition of theinvention to inhibit gene expression can be measured using a variety oftechniques including Northern blot analysis or RT-PCR for the specificmRNA or Western blot analysis for the amount of target protein present.In still another embodiment, a phenotype influenced by the amount of theprotein can be detected.

In one embodiment, the candidate iRNA agent interacts with a proteincomponent of the immune system, or has been modified not to interact,and also targets a sequence that is not involved in the immune system.

In one embodiment, the candidate iRNA agent includes at least oneribonucleotide modification (e.g., as described below), and the presenceof the modification modulates an immunostimulatory response (e.g., ascompared to what could be seen with an iRNA agent lacking themodification) when the modified iRNA agent is contacted with a cell oradministered to a subject. In preferred embodiments, one or more naturalbases of an iRNA agent are replaced by modified bases. In more preferredembodiments, one or more natural bases of an iRNA agent are replaced bymodified bases. Particular modifications to the iRNA agent may preventthe strands of the agent from separating and subsequently interactingwith the one or more protein components of the immune system. Particularmodifications may also inhibit the interaction of a double stranded orsingle stranded iRNA agent from interacting with a protein component ofthe immune system to an extent that an immune system response iseffectively prevented.

Chemical modifications can include modifications to the nucleotide base,the sugar, or the backbone. In one embodiment, the iRNA agent includes asubstitution of an adenine with a 2-substituted purine (e.g.,2-amino-adenine,), a 6-substituted purine, a 7-deaza-alkyl-substitutedpurine, a 7-deaza-alkenyl-substituted purine, a7-deaza-alkynyl-substituted purine, or a purine that is not adenine(e.g., guanine or inosine). In another embodiment, the candidate iRNAagent includes a substitution of a guanine with an inosine, anaminopurine, a 2-substituted guanine, a 7-deaza-alkyl-substitutedguanine, a 7-deaza-alkenyl-substituted guanine, a7-deaza-alkynyl-substituted guanine, or an O-6-alkylated guanine. Inanother embodiment, the candidate iRNA agent includes a substitution ofa cytosine with a 5-substituted cytosine (e.g., a 5-methyl cytosine), anN-4 substituted cytosine, a G-clamp, an analog of a G-clamp, a2-thio-cytosine, a 4-thio-cytosine, or a uracil. In one embodiment, thecandidate iRNA agent includes a substitution of a uracil with a5-substituted uracil, a 4-thio-uracil, a 5-methyl-2-thio-uracil, apseudouridine, a 1-alkylpseudouridine, a 3-alkylpseudouridine or a2-thio-uracil. In one embodiment, the iRNA agent includes a2′-deoxyfluoro, 2′-O-methyl, 2′-β-methoxyethyl, 2′-O-alkyl,2′-O-alkoxyalkyl, 2′-O-allyl, 2′-O-propyl, 2′-O-(N-methyl-acetamide(NMA), 2′-O—(N,N-dimethylaminooxyethyl), or G-clamp modification. In oneembodiment, the iRNA agent includes an arabinose-containing nucleosidethat replaces a ribonucleoside. In another embodiment, thearabinose-containing nucleoside can be a 2′-fluoroarabinose-containingnucleoside, or a 2′-O-methylarabinose-containing nucleoside. In anotherembodiment, the iRNA agent includes a deoxynucleoside that replaces aribonucleoside. In one embodiment, the deoxynucleoside is a2′-fluorodeoxynucleoside, or a 2′-O-methyldeoxynucleoside.

In one embodiment, an immunoselective iRNA agent includes at least onebackbone modification, e.g., a phosphorothioate, boronaphosphate,methylphosphonate or dithioate modification. In another embodiment, theiRNA agent includes a P-alkyl modification in the linkages between oneor more of the terminal nucleotides of an iRNA agent. In anotherembodiment, the sense and/or antisense strand is substantially free ofstereogenic phosphorus atoms having an Rp configuration, and in anotherembodiment, the sense and/or antisense strand is substantially free ofstereogenic phosphorus atoms having an Sp configuration.

In another embodiment, one or more terminal nucleotides of an iRNA agentinclude a sugar modification, e.g., a 2′ or 3′ sugar modification. Inone embodiment, the iRNA agent includes at least two sugar 2′modifications. Exemplary sugar modifications include, for example, a2′-fluoro nucleotide, a 2′-O-alkyl nucleotide, a 2′-O-alkoxyalkylnucleotide, a 2′-O-allyl nucleotide, a 2′ O-propyl nucleotide, a2′-O-methylated nucleotide (2′-O-Me), a 2′-deoxy nucleotide, a2′-deoxyfluoro nucleotide, a 2′-O-methoxyethyl nucleotide (2′-O-MOE), a2′-O-N-MeAcetamide nucleotide (2′-β-NMA), a2′-O-dimethylaminoethyloxyethyl nucleotide (2′-O-DMAEOE), a2′-aminopropyl, a 2′-hydroxy, a 2′-ara-fluoro, or 3′-amidate (3′-NH inplace of 3′-0), a locked nucleic acid (LNA), extended ethylene nucleicacid (ENA), hexose nucleic acid (HNA), or cyclohexene nucleic acid(CeNA).

In one embodiment, the iRNA agent includes a 3′ sugar modification,e.g., a 3′-O-Me modification. Preferably a 3′-O-Me modification is onthe sense strand of the iRNA agent.

In some embodiments, the iRNA agent includes a 5′-alkyl-pyrimidine e.g5′-methyl-pyrimidine (e.g., a 5′-methyl-uridine modification or a5′-methyl-cytodine modification).

The modifications described herein can be combined onto a singlecandidate iRNA agent. For example, in one embodiment, at least onenucleotide of an iRNA agent has a phosphorothioate linkage and at leastone nucleotide has a 2′ sugar modification, e.g., a 2′-O-Me or2′-deoxyfluoro modification. In another embodiment, at least onenucleotide of a candidate iRNA agent has a 5′-Me-pyrimidine and a 2′sugar modification, e.g., a 2′-deoxyfluoro or 2′-O-Me modification.

In one embodiment, the iRNA agent includes a nucleobase modification,such as a cationic modification, such as a 3′-abasic cationicmodification. The cationic modification can be e.g., an alkylamino-dT(e.g., a C6 amino-dT), an allylamino conjugate, a pyrrolidine conjugate,a pthalamido, a porphyrin, or a hydroxyprolinol conjugate.

In one embodiment, the iRNA agent includes at least one ribonucleotidemodification on the sense strand and at least one ribonucleotidemodification on the antisense strand, and the ribonucleotidemodifications on the two strands are different.

In another embodiment, the iRNA agent, e.g., an iRNA agent that caninhibit an immune response, may include a conjugate on one or morenucleotides of the iRNA agent. The conjugate can be, for example, alipophile, a terpene, a protein-binding agent, a vitamin, acarbohydrate, or a peptide. For example, the conjugate can be naproxen,nitroindole (or another conjugate that contributes to stackinginteractions), folate, ibuprofen, or a C5 pyrimidine linker. In otherembodiments, the conjugates are glyceride lipid conjugates (e.g., adialkyl glyceride derivatives), vitamin E conjugates, orthio-cholesterols.

In one embodiment, the conjugate is cholesterol, and the cholesterol isconjugated to the sense strand of the iRNA agent, e.g., by a pyrrolidinelinker, serinol linker, or hydroxyprolinol linker. In other embodiments,the conjugate is a dU-cholesterol, or cholesterol is conjugated to theiRNA agent by a disulfide linkage. In another embodiment, the conjugateis cholanic acid, and the cholanic acid is attached to the sense strandor the antisense strand. In one embodiment, the cholanic acid isattached to the sense strand and the antisense.

In another embodiment, one or more nucleotides have a 2′-5′ linkage, andpreferably, the 2′-5′ linkage is on the sense strand.

In one embodiment, the iRNA agent includes an L-sugar, preferably on thesense strand.

In one embodiment, the iRNA agent includes a methylphosphonate.

In one embodiment, the iRNA agent has been modified by replacing one ormore ribonucleotides with deoxyribonucleotides. Preferably, adjacentdeoxyribonucleotides are joined by phosphorothioate linkages, and theiRNA agent does not include more than four consecutivedeoxyribonucleotides on the sense or the antisense strands.

In some embodiments, the iRNA agent includes a difluorotoluyl (DFT)modification, e.g., 2,4-difluorotoluoyl uracil, or a guanidine toinosine substitution.

In one embodiment, the iRNA agent includes a 5′-uridine-adenine-3′(5′-UA-3′) dinucleotide wherein the uridine is a 2′-modified nucleotide,or a 5′-uridine-guanine-3′ (5′-UG-3′) dinucleotide, wherein the5′-uridine is a 2′-modified nucleotide, or a 5′-cytidine-adenine-3′(5′-CA-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modifiednucleotide, or a 5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, whereinthe 5′-uridine is a 2′-modified nucleotide, or a 5′-cytidine-cytidine-3′(5′-CC-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modifiednucleotide, or a 5′-cytidine-uridine-3′ (5′-CU-3′) dinucleotide, whereinthe 5′-cytidine is a 2′-modified nucleotide, or a 5′-uridine-cytidine-3′(5′-UC-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide. The chemically modified nucleotide in the iRNA agent may bea 2′-O-methylated nucleotide. In some embodiments, the modifiednucleotide can be a 2′-deoxy nucleotide, a 2″-deoxyfluoro nucleotide, a2′-O-methoxyethyl nucleotide, a 2′-O-NMA, a 2′-DMAEOE, a 2′-aminopropyl,2′-hydroxy, or a 2′-ara-fluoro, or 3′-amidate (3′—NH in place of 3′-O),a locked nucleic acid (LNA), extended nucleic acid (ENA), hexose nucleicacid (HNA), or cyclohexene nucleic acid (CeNA).

In one embodiment, the iRNA agent has a single overhang, e.g., one endof the iRNA agent has a 3′ or 5′ overhang and the other end of the iRNAagent is a blunt end. In another embodiment, the iRNA agent has a doubleoverhang, e.g., both ends of the iRNA agent have a 3′ or 5′ overhang,such as a dinucleotide overhang. In another embodiment, both ends of theiRNA agent have blunt ends.

In one embodiment, the iRNA agent includes a sense RNA strand and anantisense RNA strand, and the antisense RNA strand is 18-30 nucleotidesin length. In another embodiment, the iRNA agent includes a nucleotideoverhang having 1 to 4 unpaired nucleotides, which may be at the 3′-endof the antisense RNA strand, and the nucleotide overhang may have thenucleotide sequence 5′-GC-3′ or 5′-CGC-3′. The unpaired nucleotides mayhave at least one phosphorothioate dinucleotide linkage, and at leastone of the unpaired nucleotides may be chemically modified in the2′-position. In one embodiment, the double strand region of thecandidate iRNA agent includes phosphorothioate linkages on one or bothof the sense and antisense strands. In a preferred embodiment, thecandidate iRNA agent includes phosphorothioate linkages betweennucleotides 1 through 5 of the 5′ or 3′ end of the sense or antisenseagent.

In one embodiment, the antisense RNA strand and the sense RNA strand areconnected with a linker. The chemical linker may be a hexaethyleneglycol linker, a poly-(oxyphosphinico-oxy-1,3-propandiol) linker, anallyl linker, or a polyethylene glycol linker Use of a linker to connectthe antisense and sense strands, will inhibit strand separation in vivo,thereby inhibiting immuno stimulation.

In another embodiment, one or more modifications of the iRNA agent canincrease the ratio of double-stranded to single-stranded iRNA agent in abiological system (e.g., in the blood stream or in serum samples). Suchmodifications may decrease the dissociation constant (K_(D)) between thesense and antisense strands. In some embodiments, the modification thatincreases the ratio of double-stranded to single-stranded iRNA agent ina biological system is a chemical linker, such as a hexaethylene glycollinker, poly-(oxyphosphinico-oxy-1,3-propandiol) linker, allyl linker,or polyethylene glycol linker that binds the two strands together. Inone embodiment, the linker includes an ester, and the linker can becleaved by an esterase. In some embodiments, the modification is a highaffinity chemical modification, such as 2′-F, LNA, ENA, 2′-O-MOE, andC-5-propynyl pyrimidines or G-clamp and its analogs. In yet otherembodiments, the modification is a chemical crosslink, e.g., a disulfidecontaining crosslink.

In another embodiment, the immunoselective iRNA agent can include atleast two modifications. The modifications can differ from one another,and may be applied to different RNA strands of a double-stranded iRNAagent. For example, the sense strand can include at least onemodification, and the antisense strand can include a modification thatdiffers from the modification or modifications on the sense strand. Inanother example, the sense strand can include at least two differentmodifications, and the antisense strand can include at least onemodification that differs from the two different modifications on thesense strand. Accordingly, the sense strand can include multipledifferent modifications, and the antisense strand can include furthermultiple modifications, some of which are the same or unique from themodifications on the sense strand.

In one aspect, the invention relates to methods of designing an iRNAagent that binds specifically to one of the protein components of theimmune system and either inhibits or stimulates the immune cascade. Themethod may include designing an iRNA agent that includes a sense strand,and an antisense strand sufficiently complementary to hybridize to thesense strand, designing the strands such that one or more of thenucleotides on the sense and/or antisense strand are modified asdescribed herein. The iRNA agent may be further synthesized and testedin an in vitro or in vivo system for binding to a protein component ofthe immune system, e.g., as described above. For example, the iRNA agentcan be tested by assaying for an interaction with a component of theimmune system, e.g., a growth factor, such as a cytokine or interleukinprotein, or cell-surface receptor. The assay can include introducing aniRNA agent into a cell, maintaining the cell under conditions suitablefor expressing a component of the immune system, and determining whetherthe iRNA agent can interact with the component of the immune system,e.g., by co-immunoprecipitation experiments, or colocalizationexperiments, such as those facilitated by immunocytochemistrytechniques. The cell may be a mammalian cell. The iRNA agent may betested for an immune stimulatory or immune inhibitory effect in acellular assay. The iRNA agent may be tested in an in vivo system byadministering the iRNA agent to a mammal, such as a mouse, thenexamining the spleen for enlargement or increased cell proliferation, orfor an increase in production of, for example, one or more interleukinproteins, such as IL10, IL12 and/or IL6.

In some embodiments, the candidate iRNA agent is tested in a firstsystem, e.g., a cell-free system or cell-based system, and thenretested. The retest can be in the same or different assays. Forexample, the same or a different cell-based or cell-free assay can beused to confirm activity, or an animal-based system can be used toconfirm activity (e.g., gene-silencing activity, or stimulation orinhibition of the immune system) or lack of activity with respect to itseffect on an immune response. In some embodiments, the candidate iRNAagent is tested first in a cell-free or cell-based system and is thenretested in an animal-based system.

In one aspect, the invention features an iRNA agent that includes atleast one ribonucleotide modification, e.g., a ribonucleotide agentdescribed above, and the presence of the modification inhibits orstimulates an immunostimulatory response (e.g., as compared to whatcould be seen with an iRNA agent lacking the modification) when themodified iRNA agent is contacted with a cell or administered to asubject, e.g., a mammalian subject, such as a human. In preferredembodiments, one or more natural bases of an iRNA agent are replaced bymodified bases.

In one aspect the invention features a method of evaluating an iRNAagent that includes providing a candidate single stranded iRNA agenthaving at least one ribonucleotide modification; contacting thecandidate single stranded iRNA agent to a cell-free system, cell, oranimal; and evaluating the immune response in the cell-free system,cell, or animal as compared to an immune response in a cell-free system,cell, or animal that is contacted with an unmodified single strandediRNA agent. The candidate single stranded iRNA agent stimulates animmune response to a lesser or greater extent than a reference. Forexample, an unmodified iRNA agent is determined to be an iRNA agent thatmodulates an immune system response. In one embodiment, the candidatesingle-stranded iRNA agent is 15-2000 nucleotides in length (e.g., 17,19, 21, 23, 25, 27, 28, 29, 30, 100, 500, 1000, or 1500 nucleotides inlength).

In one aspect, the invention relates to an iRNA agent with dualfunction: an iRNA agent that either inhibits or stimulates the immunesystem and also sequence-specifically targets an mRNA of therapeuticrelevance for degradation via the RISC-mediated RNA interferencepathway. The immunoselective iRNA agent (e.g., an iRNA agent thatinhibits or stimulates immunostimulation) is prepared by a processdescribed herein, e.g., a process that includes providing a firstoligonucleotide that is sufficiently complementary to a target nucleicacid to anneal to the target and a second oligonucleotide sufficientlycomplementary to anneal to the first oligonucleotide. The first and/orsecond oligonucleotides include one or more modified nucleotides ornucleotide linkages described above. One or both of the antisense andsense strands, or preferably annealed antisense and sense strands, istested, e.g., as described herein, for the ability to modulate, e.g.,stimulate or inhibit an immune response, e.g., when administered to atest subject. For example, if the iRNA agent does not stimulate animmune response to a preselected magnitude, e.g., to the magnitude of aniRNA agent lacking the one or more modified nucleotides then the agentis determined to inhibit immunostimulation. The modified iRNA agent,e.g., one determined to inhibit or stimulate immunostimulation, isfurther tested to determine if it can decrease expression of a targetmRNA, e.g., by the mechanism of RNA interference, e.g., to the sameextent as the unmodified version of the iRNA agent.

In another embodiment, the invention relates to the silencing of thegene for a particular component of the immune system, e.g. toll-likereceptor 7 (TLR7) or toll-like receptor 9 (TLR9). In this embodiment,the iRNA agent is designed to be complementary to a region of the mRNAfor the component and the iRNA agent decreases the expression of thetarget through the mechanism of RNA interference.

In still another aspect, the invention relates to a pharmaceuticalcomposition including an immunoselective iRNA agent, as described above,and a pharmaceutically acceptable carrier. The pharmaceuticallyacceptable carrier may be an aqueous solution, such as phosphatebuffered saline, or it may include a micellar structure, such as aliposome, capsid, capsoid, polymeric nanocapsule, or polymericmicrocapsule.

In yet a further aspect, the invention relates to a method for treatinga disease or disorder in a subject. The method includes identifying asubject having or at risk for developing the disease, administering apharmaceutical composition containing an immunoselective iRNA agenthaving one or more of the modified nucleotides or linkages describedabove, and a pharmaceutically acceptable carrier. The subject may bemonitored for an effect on the immune system, e.g., an immunostimulatoryor immunoinhibitory response, such as by monitoring for increasedexpression of a growth factor, such as a cytokine or a cell-surfacereceptor (e.g., a Toll-like receptor) as described above. Cytokines ofinterest can be those expressed from T cells, B cells, monocytes,macrophages, dendritic cells, or natural killer cells of the subject.The assays can be performed using blood or serum samples from thesubject. The disease or disorder can be one where it is particularlyundesirable to stimulate the immune system, e.g., in a patient that hasreceived organ, tissue or bone marrow transplants. In anotheralternative, the disease or disorder can be one where it is particularlydesirable to stimulate the immune system, e.g., in patients with canceror viral diseases. In one embodiment, the subject is immunocompromised,and an iRNA agent that includes nucleotide modifications stimulates animmune response in a cell to a greater extent than an iRNA agent thatdoes not include nucleotide modifications. The subject may be a mammal,such as a human.

In a preferred embodiment, administration of an immunoselective iRNAagent is for treatment of a disease or disorder present in the subject.In another preferred embodiment, administration of the iRNA agent is forprophylactic treatment.

It is therefore an object of the present invention to providepolynucleotides/oligonucleotides which are capable of stimulating ananti-viral response, in particular, a type I IFN response. It is anotherobject of the present invention to provide a pharmaceutical compositioncapable of inducing an anti-viral response, in particular, type I IFNproduction, in a patient for the prevention and treatment of diseasesand disorders such as viral infection. It is also an object of thepresent invention to provide a pharmaceutical composition for treatingtumor.

The disease and/or disorder include, but are not limited to infections,tumor, allergy, multiple sclerosis, and immune disorders.

Infections include, but are not limited to, viral infections, bacterialinfections, anthrax, parasitic infections, fungal infections and prioninfection. Viral infections include, but are not limited to, infectionby hepatitis C, hepatitis B, herpes simplex virus (HSV), HIV-AIDS,poliovirus, encephalomyocarditis virus (EMCV) and smallpox virus.Examples of (+) strand RNA viruses which can be targeted for inhibitioninclude, without limitation, picornaviruses, caliciviruses, nodaviruses,coronaviruses, arteriviruses, flaviviruses, and togaviruses. Examples ofpicornaviruses include enterovirus (poliovirus 1), rhinovirus (humanrhinovirus 1A), hepatovirus (hepatitis A virus), cardiovirus(encephalomyocarditis virus), aphthovirus (foot-and-mouth disease virusO), and parechovirus (human echovirus 22). Examples of calicivirusesinclude vesiculovirus (swine vesicular exanthema virus), lagovirus(rabbit hemorrhagic disease virus), “Norwalk-like viruses” (Norwalkvirus), “Sapporo-like viruses” (Sapporo virus), and “hepatitis E-likeviruses” (hepatitis E virus). Betanodavirus (striped jack nervousnecrosis virus) is the representative nodavirus. Coronaviruses includecoronavirus (avian infections bronchitis virus) and torovirus (Bernevirus). Arterivirus (equine arteritis virus) is the representativearteriviridus. Togavirises include alphavirus (Sindbis virus) andrubivirus (Rubella virus). Finally, the flaviviruses include flavivirus(Yellow fever virus), pestivirus (bovine diarrhea virus), andhepacivirus (hepatitis C virus).

In certain embodiments, the viral infections are selected from chronichepatitis B, chronic hepatitis C, HIV infection, RSV infection, HSVinfection, VSV infection, CMV infection, and influenza infection.

In one embodiment, the infection to be prevented and/or treated is upperrespiratory tract infections caused by viruses and/or bacteria. Inanother embodiment, the infection to be prevented and/or treated is birdflu.

Bacterial infections include, but are not limited to, streptococci,staphylococci, E. coli, pseudomonas.

In one embodiment, bacterial infection is intracellular bacterialinfection. Intracellular bacterial infection refers to infection byintracellular bacteria such as mycobacteria (tuberculosis), chlamydia,mycoplasma, listeria, and facultative intracellular bacteria such asstaphylococcus aureus.

Parasitic infections include, but are not limited to, worm infections,in particular, intestinal worm infection.

Tumors include both benign and malignant tumors (i.e., cancer).

Cancers include, but are not limited to biliary tract cancer, braincancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer,endometrial cancer, esophageal cancer, gastric cancer, intraepithelialneoplasm, leukemia, lymphoma, liver cancer, lung cancer, melanoma,myelomas, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer,prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer,thyroid cancer and renal cancer.

In certain embodiments, cancers are selected from hairy cell leukemia,chronic myelogenous leukemia, cutaneous T-cell leukemia, chronic myeloidleukemia, non-Hodgkin's lymphoma, multiple myeloma, follicular lymphoma,malignant melanoma, squamous cell carcinoma, renal cell carcinoma,prostate carcinoma, bladder cell carcinoma, breast carcinoma, ovariancarcinoma, non-small cell lung cancer, small cell lung cancer,hepatocellular carcinoma, basaliom, colon carcinoma, cervical dysplasia,and Kaposi's sarcoma (AIDS-related and non-AIDS related).

Allergies include, but are not limited to, respiratory allergies,contact allergies and food allergies.

Immune disorders include, but are not limited to, autoimmune diseases,immunodeficiency, and immunosuppression.

Autoimmune diseases include, but are not limited to, diabetes mellitus,arthritis (including rheumatoid arthritis, juvenile rheumatoidarthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis,encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's disease,aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerativecolitis, asthma, allergic asthma, cutaneous lupus erythematosus,scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversalreactions, erythema nodosum leprosum, autoimmune uveitis, allergicencephalomyelitis, acute necrotizing hemorrhagic encephalopathy,idiopathic bilateral progressive sensorineural hearing, loss, aplasticanemia, pure red cell anemia, idiopathic thrombocytopenia,polychondritis, Wegener's granulomatosis, chronic active hepatitis,Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves'disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, andinterstitial lung fibrosis.

Immunodeficiencies include, but are not limited to, spontaneousimmunodeficiency, acquired immunodeficiency (including AIDS),drug-induced immunodeficiency (such as that induced byimmunosuppressants used in transplantation and chemotherapeutic agentsused for treating cancer), immunosuppression caused by chronichemodialysis, trauma or surgical procedures.

Immunosuppression includes, but is not limited to, bone marrowsuppression by cytotoxic chemotherapy.

In one embodiment, the pharmaceutical composition is a tumor vaccine.The iRNA agent described in the invention or the bacterial RNA mayinduce tumor cell apoptosis through binding to RIG-I, induce type I IFN₁IL-18 and/or IL-1β production by the tumor cells, directly and/orindirectly activate effector cells of innate immunity such as NK cells,NKT cells, and γδ T cells, and/or directly and/or indirectly inactivatesuppressor T cells, thereby leading to tumor cell growth inhibitionand/or destruction.

Tumor cells which have been stimulated with an RIG-I ligand, such as theiRNA agent described in the present invention or a bacterial RNA, mayalso be used as a tumor vaccine.

In a preferred embodiment, the RNA oligonucleotide is a single-strandedRNA oligonucleotide which does not contain any sequence which is capableof forming any intramolecular or intermolecular double-strandedstructure with itself under physiological condition, in particular,physiological condition inside a cell, and the nucleotide sequence ofthe ssRNA is complentary to a disease/disorder-related RNA.

In one embodiment, the disease/disorder-related RNA is an mRNA of adisease/disorder-related gene. In another embodiment, thedisease/disorder-related RNA is a miRNA. The disease/disorder-relatedRNA may be a endogenous cellular RNA, a viral RNA, a RNA from aninvading microorganism or organism such as a bacterium, a fungus, or aparasite.

The degree of complementarity is preferably at least 50%, 60%, 70%, morepreferably at least 75%, 80%, 85%, 90%, even more preferably at least95%, 96%, 97%, 98%, 99%, and most preferably 100%.

In one aspect, the invention features a method of increasing the ratioof double stranded iRNA (dsiRNA) agent to single stranded iRNA (ssiRNA)agent in a human by administering to the human a dsiRNA agent thatincludes one or more modifications that inhibit disassociation of thedsiRNA agent as compared to a dsiRNA agent that does not include themodifications. In one embodiment, the modifications include a chemicallinker, such as a hexaethylene glycol linker,poly(oxyphosphinico-oxy-1,3-propandiol) linker, allyl linker, orpolyethylene glycol linker. In another embodiment, the modificationsincrease the melting temperature of the dsiRNA as compared to a dsiRNAthat does not include the modifications. Such modifications can includea locked nucleic acid, G-clamp, 2′-O-methyl, 2′-fluoro,2′-O-methoxyethyl, 2-thio-pyrimidine, 2-amino-adenine or pseudouridine.In some embodiments, the modifications of the dsiRNA agent occur only inthe sense strand of the dsiRNA agent or only in the antisense strand ofthe dsiRNA agent.

In one aspect, the invention features a method of selecting a patientsuitable for treatment with an immunoselective iRNA agent describedherein. In one embodiment, the selection of the patient is based on needfor either immunostimulation or immunosuppression. In anotherembodiment, selection is based on the identification of a patient inneed of decreased expression of a gene not involved in the immunesystem, and also in need of increased or decreased immune systemfunction. The patient's need with respect to immune system function willdetermine which modifications will be incorporated into the therapeuticimmunoselective iRNA agent.

In one aspect, the invention features an immunoselective iRNA agent thatstimulates the immune system. For example, a stimulatory immunoselectiveiRNA agent includes one or more modifications, e.g., modificationsdescribed herein for stimulating the immune system. For example, themodified iRNA agent will stimulate the immune system to a greater extentthan an iRNA agent that does not include the one or more modifications.These stimulatory immunoselective iRNA agents can be administered toimmunocompromised patients, or can be administered with a second therapyto off-set immunocompromising effects of the second therapy. The secondtherapeutic can be a chemotherapy or antibiotic, for example. Astimulatory immunoselective iRNA agent can be administered to a subjecthaving or at risk for developing a tumor, an autoimmune disease, airwayinflammation (e.g., asthma), an allergy (e.g., a food or respiratoryallergy), or a pathogenic disease, such as a disease caused by abacteria, fungus, parasite, virus or viroid particle, or prion.

DEFINITIONS

The term “linker” or “spacer” generally refers to any moiety that can beattached to an oligoribonucleotide by way of covalent or non-covalentbonding through a sugar, a base, or the backbone. The linker/spacer canbe used to attach two or more nucleosides or can be attached to the 5′and/or 3′ terminal nucleotide in the oligoribonucleotide. Such linkercan be either a non-nucleotidic linker or a nucleotidic linker.

The term “non-nucleotidic linker” generally refers to a chemical moietyother than a nucleotidic linkage that can be attached to anoligoribonucleotide by way of covalent or non-covalent bonding.Preferably such non-nucleotidic linker is from about 2 angstroms toabout 200 angstroms in length, and may be either in a cis or transorientation, (e.g. d(T)_(n); wherein n is 1-10) or non-nucleotidic (forexample a linker described herein, e.g. optionally substituted alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl or heteroaryl).

The term “nucleotidic linkage” generally refers to a chemical linkage tojoin two nucleosides through their sugars (e.g. 3′-3′, 2′-3′,2′-5′,3′-5′) consisting of a phosphate, non-phosphate, charged, or neutralgroup (e.g., phosphodiester, phosphorothioate, phosphorodithioate,alkylphosphonate (e.g. methylphosphonate), amide, ester, disulfide,thioether, oxime and hydrazone linkage between adjacent nucleosides.

In one embodiment, the linker/spacer between the two oligonucleotidescomprises a cleavable linking group, for example a group that ispotentially biodegradable by enzymes present in the organism such asnucleases and proteases or cleavable at acidic pH or under reductiveconditions, such as by glutathione present at high levelsintracelullarly. Some exemplary cleavable linking groups include, butare not limited to, disulfides, amides, esters, peptide linkages andphosphodiesters. Copending U.S. application Ser. No. 10/985,426, filedNov. 9, 2004, describes cleavable tethers that are amenable for use asspacers comprising cleavable groups.

The cleavable linking group can be internal to the spacer or may bepresent at one or both terminal ends of the spacer. In one embodiment,the cleavable linking group is between one of the oligonucleotides andthe spacer. In one embodiment, the cleavable linking group is present onboth ends of the spacer. In one embodiment, the cleavable linking groupis internal to the spacer.

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine.

The term “aliphatic” refers to non-aromatic moiety that may contain anycombination of carbon atoms, hydrogen atoms, halogen atoms, oxygen,nitrogen or other atoms, and optionally contain one or more units ofunsaturation, e.g., double and/or triple bonds. An aliphatic group maybe straight chained, branched or cyclic and preferably contains betweenabout 1 and about 24 carbon atoms, more typically between about 1 andabout 12 carbon atoms. In addition to aliphatic hydrocarbon groups,aliphatic groups include, for example, polyalkoxyalkyls, such aspolyalkylene glycols, polyamines, and polyimines, for example. Suchaliphatic groups may be further substituted.

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁-C₁₂ alkyl indicates that the group may have from1 to 12 (inclusive) carbon atoms in it. The term “haloalkyl” refers toan alkyl in which one or more hydrogen atoms are replaced by halo, andincludes alkyl moieties in which all hydrogens have been replaced byhalo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may beoptionally inserted with O, N, or S. The terms “aralkyl” refers to analkyl moiety in which an alkyl hydrogen atom is replaced by an arylgroup. Aralkyl includes groups in which more than one hydrogen atom hasbeen replaced by an aryl group. Examples of “aralkyl” include benzyl,9-fluorenyl, benzhydryl, and trityl groups.

The term “alkenyl” refers to a straight or branched hydrocarbon chaincontaining 2-8 carbon atoms and characterized in having one or moredouble bonds. Examples of a typical alkenyl include, but not limited to,allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. The term“alkynyl” refers to a straight or branched hydrocarbon chain containing2-8 carbon atoms and characterized in having one or more triple bonds.Some examples of a typical alkynyl are ethynyl, 2-propynyl, and3-methylbutynyl, and propargyl. The sp² and sp³ carbons may optionallyserve as the point of attachment of the alkenyl and alkynyl groups,respectively.

The terms “alkylamino” and “dialkylamino” refer to —NH(alkyl) and —N(alkyl)₂ radicals respectively. The term “aralkylamino” refers to a—NH(aralkyl) radical. The term “alkoxy” refers to an —O-alkyl radical,and the terms “cycloalkoxy” and “aralkoxy” refer to an —O-cycloalkyl andO-aralkyl radicals respectively. The term “siloxy” refers to a R₃SiO—radical. The term “mercapto” refers to an SH radical. The term“thioalkoxy” refers to an —S-alkyl radical.

The term “alkylene” refers to a divalent alkyl (i.e., —R—), e.g., —CH₂—,—CH₂CH₂—, and —CH₂CH₂CH₂—. The term “alkylenedioxo” refers to a divalentspecies of the structure —O—R—O—, in which R represents an alkylene.

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system, wherein any ring atom can be substituted.Examples of aryl moieties include, but are not limited to, phenyl,naphthyl, anthracenyl, and pyrenyl.

The term “cycloalkyl” as employed herein includes saturated cyclic,bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12carbons, wherein any ring atom can be substituted. The cycloalkyl groupsherein described may also contain fused rings. Fused rings are ringsthat share a common carbon-carbon bond or a common carbon atom (e.g.,spiro-fused rings). Examples of cycloalkyl moieties include, but are notlimited to, cyclohexyl, adamantyl, and norbornyl.

The term “heterocyclyl” refers to a nonaromatic 3-10 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively), whereinany ring atom can be substituted. The heterocyclyl groups hereindescribed may also contain fused rings. Fused rings are rings that sharea common carbon-carbon bond or a common carbon atom (e.g., spiro-fusedrings). Examples of heterocyclyl include, but are not limited totetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino,pyrrolinyl and pyrrolidinyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein any ring atomcan be substituted. The heteroaryl groups herein described may alsocontain fused rings that share a common carbon-carbon bond.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl,cycloalkenyl, aryl, or heteroaryl group at any atom of that group.Suitable substituents include, without limitation, alkyl, alkenyl,alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO₃H, sulfate,phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy,ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl),S(O)_(n)alkyl (where n is 0-2), S(O)_(n) aryl (where n is 0-2), S(O)_(n)heteroaryl (where n is 0-2), S(O)_(n) heterocyclyl (where n is 0-2),amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, andcombinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide(mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof),sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinationsthereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstitutedheterocyclyl, and unsubstituted cycloalkyl. In one aspect, thesubstituents on a group are independently any one single, or any subsetof the aforementioned substituents.

A “protected” moiety refers to a reactive functional group, e.g., ahydroxyl group or an amino group, or a class of molecules, e.g., sugars,having one or more functional groups, in which the reactivity of thefunctional group is temporarily blocked by the presence of an attachedprotecting group. Protecting groups useful for the monomers and methodsdescribed herein can be found, e.g., in Greene, T. W., Protective Groupsin Organic Synthesis (John Wiley and Sons: New York), 1981, which ishereby incorporated by reference.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl)phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the oligomeric compounds of theinvention: i.e., salts that retain the desired biological activity ofthe parent compound and do not impart undesired toxicological effectsthereto. For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

Ligand

In the present invention, the ligand is an aromatic group, aralkylgroup, or the radical of a steroid, bile acid, lipid, folic acid,pyridoxal, B12, riboflavin, biotin, polycyclic compound, crown ether,intercalator, cleaver molecule, protein-binding agent, carbohydrate, oran optionally substituted saturated 5-membered ring. In certaininstances, the ligand is an aralkyl group, e.g., a 2-arylpropanoylmoiety. The structural features of the ligand are selected so that theligand will bind to at least one protein in vivo. In certainembodiments, the structural features of the ligand are selected so thatligand binds to serum, vascular, or cellular proteins. In certainembodiments, the structural features of the ligand promote binding toalbumin, an immunoglobulin, a lipoprotein, α-2-macroglubulin, orα-1-glycoprotein.

A large number of steroids are known in the art and are amenable to thepresent invention. Representative examples of steriods includecholesterol, 5β-cholanic acid, progesterone, aldosterone,dehydroaldosterone, isoandrosterone, esterone, estradiol, ergosterol,dehydroergosterol, lanosterol, 4-cholesten-3-one, guggulsterone,testosterone, nortestosterone, formestane, hydroxyecdysone, ketoestriol,corticosterone, dienestrol, dihydroxypregnanone, pregnanone, copornmon,equilenin, equilin, estriol, ethinylestradiol, mestranol, moxestrol,mytatrienediol, quinestradiol, quinestrol, helvolic acid, protostadiene,fusidic acid, cycloartenol, tricallol, cucurbitanin cedrelone, euphol,dammerenediol, parkeol, dexametasone, methylprednisolone, prednisolone,hydrocortisone, parametasone, betametasone, cortisone, fluocinonide,fluorometholone, halcinonide, and budesonide, or any one of them furthersubstituted with one or more of hydroxyl, halogen, amino, alkylamino,alkyl, carboxylic acid, ester, amide, carbonyl, alkoxyl, or cyano.

A large number of bile acids are known in the art and are amenable tothe present invention. Bile acids occur in conjugation with glycine ortaurine in bile of most vertebrates and some of them find use inmedicine. Thus, some bile acids—due to their inherent pharmacologicalproperties—are used as cholerectics (see, for example, James E. F.Reynolds (editor) Martindale The Extra Pharmacopoeia, 30^(th) Edition,The Pharmaceutical Press, London (1993), page 1341). Representativeexamples of bile acids include cholic acid, deoxycholic acid,taurocholic acid, glycocholic acid, glycodeoxycholic acid,taurodeoxycholic acid, ursodeoxycholic acid, and chenodeoxycholic acid.Additional bile acids amenable to the present invention include thosedescribed in U.S. Pat. Nos. 5,641,767; 5,656,277; 5,610,151; 5,428,182;and 3,910,888.

A large number of lipids are known in the art and are amenable to thepresent invention. Representative examples of lipids include lauricacid, myristic acid, palmitic acid, stearic acid, arachidic acid,palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonicacid, triacylglycerols, phosphoacylglycerols, sphingolipids,monoterpenes, sesquiterpenes, diterpenes, sesterterpenes, triterpenes,and tetraterpenes.

A large number of aromatic compounds are known in the art and areamenable to the present invention. Representative examples of aromaticcompounds include optionally substituted phenyl, naphthyl, anthracenyl,phenanthrenyl, pyrenyl, pyridinyl, quinolinyl, acridinyl,phenathridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinoxalinyl,quinazolinyl, 1,7-phenanthrolinyl, indolyl, thianaphthenyl,benzoxazolyl, benzofuranyl, 1,2-benzisoxazolyl, benzimidazolyl,pyrrolyl, thiophenyl, isoxazolyl, pyrazolyl, thiazolyl, imidazolyl,tetrazolyl, and furanyl.

A large number of carbohydrates are known in the art and are amenable tothe present invention. Representative examples of carbohydrates includeerythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose,glucose, mannose, gulose, idose, galactose, and talose; or adisaccharide or trisaccharide formed via a 1,4 glycoside linkage betweenany of them. In certain instances, the carbohydrate is a hexose orpentose.

A large number of polycyclic compounds are known in the art and areamenable to the present invention. Representative classes of polycycliccompounds include bicyclic compounds wherein, the first and second ringare independently a 3, 4, 5, or 6-member saturated or unsaturated carbonring containing 0, 1, 2, or 3 hetereoatoms selected from the groupconsisting of O, N, or S. In certain instances, the first ring is anaromatic ring. In certain instances, the second ring is an aromaticring. In certain instances, both rings are saturated. In certaininstances, the first ring contains no heteroatoms. In certain instances,the second ring contains to heteroatoms. In certain instances, the firstring contains a nitrogen atom. In certain instances, the second ringcontains a nitrogen atom. In certain instances, the polycyclic compoundis a tricyclic compound, wherein the first, second, and third ring areindependently a 3, 4, 5, or 6-member saturated or unsaturated carbonring containing 0, 1, 2, or 3 hetereoatoms selected from the groupconsisting of O, N, or S. In certain instances, the first ring is anaromatic ring. In certain instances, the second ring is an aromaticring. In certain instances, the third ring is an aromatic ring. Incertain instances, all three rings are saturated. In certain instances,the first ring contains no heteroatoms. In certain instances, the secondring contains to heteroatoms. In certain instances, the third ringcontains to heteroatoms. In certain instances, the first ring contains anitrogen atom. In certain instances, the second ring contains a nitrogenatom. In certain instances, the third ring contains a nitrogen atom. Incertain instances, the polycyclic compound is a bridged polycycliccompound. In certain instances, the polycyclic compound is abicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.1]octane,bicyclo[3.2.2]nonane, or bicyclo[3.3.1]nonane.

A large number of crown ethers are known in the art and are amenable tothe present invention. Crown ethers are macrocyclic, polyether, neutralcompounds containing 4-20 oxygen atoms each separated from the next bytwo or more carbon atoms. Macrocyclic polyethers have been found to formstable complexes with salts of alkali metals and other metals andammonium salts; “Macrocyclic polyethers and their complexes”, C. J.Pederson et al, Angew. Chem. Intern. Ed., Vol. 11, page 16, (1972) andU.S. Pat. Nos. 3,562,295 and 3,687,978. Since the stereo models ofmacrocyclic polyethers give a crown-like appearance, they are commonlydesignated as N-crown-M polyethers, wherein N is the total number ofatoms in the polyether ring and M is the number of oxygen atoms in thepolyether ring. Crown polyethers ranging in size from cyclic tetramersof ethylene oxide ([12]-crown-4) and propylene oxide ([16]-crown-4) to60-membered polyether rings (dibenzo[60]-crown-20) have been reported.Preferred crown ethers include 12-crown-4, 15-crown-5, and 18-crown-6.

A large number of oligonucleotide intercalators are known in the art andare amenable to the present invention. One class of intercalators areDNA intercalators which bind noncovalently to duplex DNA and arecharacterized by a flat molecule which inserts between base pairs of thedouble helix of DNA. Representative examples of intercalators includep-carboxy methidium, p-carboxy ethidium, acridine and ellipticine.

A large number of oligonucleotide cleaver molecules are known in the artand are amenable to the present invention. A cleaver molecule is acompound that can sever an oligonucleotide strand. Bleomycin, aglycopeptide antibiotic, is known to bind to and cleave DNA in areaction that depends on the presence of ferrous ion and molecularoxygen, “Bleomycin: Chemical, Biochemical and Biological Aspects”;Hecht, S. M., Ed.; Springer Verlag New York, 1979; Sausville, E. A.;Peisach, J.; Horwitz, S. B. “Biochemistry” 1978, 17, 2740. Burger, R.M.; Peisach, J; Horwitz, S. B. “Life Sciences” 1981, 28, 715; and Lown,J. W.; Sim, S. F. “Biochem. Biophys. Res. Comm.” 1977, 77, 1150. Theantitumor agent streptonigrin is also capable of causing single strandbreaks in DNA using oxygen and cuprous ion, Cone, R; Hasan, S. K.; Lown,J. W.; Morgan, A. R. “Can. J. Biochem.” 1976, 54, 219. Recently, the1-10 phenanthroline-cuprous complex has been shown to cleave DNA in thepresence of oxygen, Sigman, D. S.; Graham, D. R.; D'Aurora, V.; Stern,A. M. “J. Biol. Chem.” 1979, 254, 12269; Graham, D. R.; Marshall, L. E.;Reich, K. A.; Sigman, D. S. “J. Amer. Chem. Soc.” 1980, 102, 5419;Marshall, L. E.; Graham, D. R.; Reich, K. A.; Sigman, D. S.“Biochemistry” 1981, 20, 244; and Que, B. G.; Downey, K. M.; So., A. G.“Biochemistry” 1980, 19, 5987. In addition, methidium, ethidium, andcisplatin are known to cleave oligonucleotide sequences.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; nuclease-resistanceconferring moieties; and natural or unusual nucleobases. Generalexamples include lipophiles, lipids, steroids (e.g., cholesterol, uvaol,hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),vitamins (e.g., folic acid, vitamin A, vitamin E, biotin, pyridoxal),carbohydrates, proteins, protein binding agents, integrin targetingmolecules, polycationics, peptides, polyamines, and peptide mimics.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

iRNA Agent Structure

The monomers described herein can be used to make oligonucleotides whichare useful as iRNA agents, e.g., RNA molecules, (double-stranded;single-stranded) that mediate RNAi, e.g., with respect to an endogenousgene of a subject or to a gene of a pathogen. In most cases the iRNAagent will incorporate monomers described herein together with naturallyoccurring nucleosides or nucleotides or with other modified nucleosidesor nucleotides. The modified monomers can be present at any position inthe iRNA agent, e.g., at the terminii or in the middle region of an iRNAagent or in a duplex region or in an unpaired region. In a preferredembodiment iRNA agent can have any architecture, e.g., architecturedescribed herein. E.g., it can be incorporated into an iRNA agent havingan overhang structure, a hairpin or other single strand structure or atwo-strand structure, as described herein.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, ornucleoside surrogate, all of which are defined herein (see, e.g., thesection below entitled RNA Agents). While numerous modified RNAs andnucleoside surrogates are described, preferred examples include thosewhich have greater resistance to nuclease degradation than do unmodifiedRNAs. Preferred examples include those which have a 2′ sugarmodification, a modification in a single strand overhang, preferably a3′ single strand overhang, or, particularly if single stranded, a 5′modification which includes one or more phosphate groups or one or moreanalogs of a phosphate group.

An “iRNA agent,” as used herein, is an RNA agent which can, or which canbe cleaved into an RNA agent which can, stimulate or inhibit an immuneresponse, or have no effect on an immune response. An iRNA agent mayalso down regulate the expression of a target gene, preferably anendogenous or pathogen target RNA. While not wishing to be bound bytheory, an iRNA agent that down regulates expression of a target genemay act by one or more of a number of mechanisms, includingpost-transcriptional cleavage of a target mRNA (sometimes referred to inthe art as RNAi), or pre-transcriptional or pre-translationalmechanisms. An iRNA agent can include a single strand or can includemore than one strands, e.g., it can be a double stranded iRNA agent. Ifthe iRNA agent is a single strand it is particularly preferred that itinclude a 5′ modification which includes one or more phosphate groups orone or more analogs of a phosphate group.

For ease of exposition the term nucleotide or ribonucleotide issometimes used herein in reference to one or more monomeric subunits ofan RNA agent. It will be understood herein that the usage of the term“ribonucleotide” or “nucleotide” can, in the case of a modified RNA ornucleotide surrogate, also refer to a modified nucleotide, or surrogatereplacement moiety at one or more positions.

As discussed elsewhere herein, an iRNA agent will often be modified orinclude nucleoside surrogates in addition to the ribose replacementmodification subunit (RRMS). Single stranded regions of an iRNA agentwill often be modified or include nucleoside surrogates, e.g., theunpaired region or regions of a hairpin structure, e.g., a region whichlinks two complementary regions, can have modifications or nucleosidesurrogates. Modification to stabilize one or more 3′- or 5′-terminus ofan iRNA agent, e.g., against exonucleases, or to favor the antisensesRNA agent to enter into RISC are also favored. Modifications caninclude C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyllinkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethyleneglycol, hexaethylene glycol), special biotin or fluorescein reagentsthat come as phosphoramidites and that have another DMT-protectedhydroxyl group, allowing multiple couplings during RNA synthesis.

The Bases. Adenine, guanine, cytosine and uracil are the most commonbases found in RNA. These bases can be modified or replaced to provideRNA's having improved properties. E.g., nuclease resistantoligoribonucleotides can be prepared with these bases or with syntheticand natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine,nubularine, isoguanisine, or tubercidine) and any one of the abovemodifications. Alternatively, substituted or modified analogs of any ofthe above bases, e.g., “unusual bases” and “universal bases” describedherein, can be employed. Examples include without limitation2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo,amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines andguanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, N6, N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases. Further purines and pyrimidines include those disclosed in U.S.Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613.

A universal base can form base pairs with each of the natural DNA/RNAbases, exhibiting relatively little discrimination between them. Ingeneral, the universal bases are non-hydrogen bonding, hydrophobic,aromatic moieties which can stabilize e.g., duplex RNA or RNA-likemolecules, via stacking interactions. A universal base can also includehydrogen bonding substituents. As used herein, a “universal base” caninclude anthracenes, pyrenes or any one of the following:

Generally, base changes are less preferred for promoting stability, butthey can be useful for other reasons, e.g., some, e.g.,2,6-diaminopurine and 2 amino purine, are fluorescent. Modified basescan reduce target specificity. This should be taken into considerationin the design of iRNA agents.

General References. The oligoribonucleotides and oligoribonucleosidesused in accordance with this invention may be with solid phasesynthesis, see for example “Oligonucleotide synthesis, a practicalapproach”, Ed. M. J. Gait, IRL Press, 1984; “Oligonucleotides andAnalogues, A Practical Approach”, Ed. F. Eckstein, IRL Press, 1991(especially Chapter 1, Modern machine-aided methods ofoligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotidesynthesis, Chapter 3,2′-O-Methyloligoribonucleotide-s: synthesis andapplications, Chapter 4, Phosphorothioate oligonucleotides, Chapter 5,Synthesis of oligonucleotide phosphorodithioates, Chapter 6, Synthesisof oligo-2′-deoxyribonucleoside methylphosphonates, and Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein.

Modification described in WO 00/44895, WO01/75164, or WO02/44321 can beused herein.

The disclosure of all publications, patents, and published patentapplications listed herein are hereby incorporated by reference.

Phosphate Group References. The preparation of phosphinateoligoribonucleotides is described in U.S. Pat. No. 5,508,270. Thepreparation of alkyl phosphonate oligoribonucleotides is described inU.S. Pat. No. 4,469,863. The preparation of phosphoramiditeoligoribonucleotides is described in U.S. Pat. No. 5,256,775 or U.S.Pat. No. 5,366,878. The preparation of phosphotriesteroligoribonucleotides is described in U.S. Pat. No. 5,023,243. Thepreparation of borano phosphate oligoribonucleotide is described in U.S.Pat. Nos. 5,130,302 and 5,177,198. The preparation of 3′-Deoxy-3′-aminophosphoramidate oligoribonucleotides is described in U.S. Pat. No.5,476,925. 3′-Deoxy-3′-methylenephosphonate oligoribonucleotides isdescribed in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801.Preparation of sulfur bridged nucleotides is described in Sproat et al.Nucleosides Nucleotides 1988, 7,651 and Crosstick et al. TetrahedronLett. 1989, 30, 4693.

Sugar Group References. Modifications to the 2′ modifications can befound in Manoharan, Biochimica et Biophysica Acta 1489:117-130, 1999;Verma, S. et al. Annu. Rev. Biochem. 67:99-134, 1998 and referencestherein. Specific modifications to the ribose can be found in thefollowing references: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993,36, 831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938),“LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310). iRNA-specificchemical modifications are described in Manoharan, Current Opinion inChemical Biology 8:570-579, 2004.

Cationic Groups

Modifications can also include attachment of one or more cationic groupsto the sugar, base, and/or the phosphorus atom of a phosphate ormodified phosphate backbone moiety. A cationic group can be attached toany atom capable of substitution on a natural, unusual or universalbase. A preferred position is one that does not interfere withhybridization, i.e., does not interfere with the hydrogen bondinginteractions needed for base pairing. A cationic group can be attachede.g., through the C2′ position of a sugar or analogous position in acyclic or acyclic sugar surrogate. Cationic groups can include e.g.,protonated amino groups, derived from e.g., O-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino);aminoalkoxy, e.g., O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, or amino acid); orNH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino).

Nonphosphate Linkages

Modifications can also include the incorporation of nonphosphatelinkages at the 5′ and/or 3′ end of a strand. Examples of nonphosphatelinkages which can replace the phosphate group include methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl,carbamate, amide, thioether, ethylene oxide linker, sulfonate,sulfonamide, thioformacetal, formacetal, oxime, methyleneimino,methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo andmethyleneoxymethylimino. Preferred replacements include the methylphosphonate and hydroxylamino groups.

Modifications can also include linking two sugars via a phosphate ormodified phosphate group through the 2′ position of a first sugar andthe 5′ position of a second sugar. Also contemplated are invertedlinkages in which both a first and second sugar are eached linkedthrough the respective3′ positions. Modified RNA's can also include“abasic” sugars, which lack a nucleobase at C-1′. The sugar group canalso contain one or more carbons that possess the oppositestereochemical configuration than that of the corresponding carbon inribose. Thus, a modified iRNA agent can include nucleotides containinge.g., arabinose, as the sugar. In another subset of this modification,the natural, unusual, or universal base may have the α-configuration.Modifcations can also include L-RNA.

Modifications can also include 5′-phosphonates, e.g.,P(O)(O⁻)₂—X—C^(5′)-sugar (X═CH₂, CF₂, CHF and 5′-phosphate prodrugs,e.g., P(O)[OCH2CH2SC(O)R]₂CH₂C^(5′)-sugar. In the latter case, theprodrug groups may be decomposed via reaction first with carboxyesterases. The remaining ethyl thiolate group via intramolecular S_(N)2displacement can depart as episulfide to afford the underivatizedphosphate group.

Modification can also include the addition of conjugating groupsdescribed elsewhere herein, which are preferably attached to an iRNAagent through any amino group available for conjugation.

Nuclease resistant modifications include some which can be placed onlyat the terminus and others which can go at any position. Generally themodifications that can inhibit hybridization so it is preferably to usethem only in terminal regions, and preferable to not use them at thecleavage site or in the cleavage region of an sequence which targets asubject sequence or gene. The can be used anywhere in a sense sequence,provided that sufficient hybridization between the two sequences of theiRNA agent is maintained. In some embodiments it is desirable to put theNRM at the cleavage site or in the cleavage region of a sequence whichdoes not target a subject sequence or gene, as it can minimizeoff-target silencing.

In addition, an iRNA agent described herein can have an overhang whichdoes not form a duplex structure with the other sequence of the iRNAagent—it is an overhang, but it does hybridize, either with itself, orwith another nucleic acid, other than the other sequence of the iRNAagent.

In most cases, the nuclease-resistance promoting modifications will bedistributed differently depending on whether the sequence will target asequence in the subject (often referred to as an anti-sense sequence) orwill not target a sequence in the subject (often referred to as a sensesequence). If a sequence is to target a sequence in the subject,modifications which interfere with or inhibit endonuclease cleavageshould not be inserted in the region which is subject to RISC mediatedcleavage, e.g., the cleavage site or the cleavage region (As describedin Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated byreference, cleavage of the target occurs about in the middle of a 20 or21 nt guide RNA, or about 10 or 11 nucleotides upstream of the firstnucleotide which is complementary to the guide sequence. As used hereincleavage site refers to the nucleotide on either side of the cleavagesite, on the target or on the iRNA agent strand which hybridizes to it.Cleavage region means an nucleotide with 1, 2, or 3 nucleotides of thecleave site, in either direction.)

Such modifications can be introduced into the terminal regions, e.g., atthe terminal position or with 2, 3, 4, or 5 positions of the terminus,of a sequence which targets or a sequence which does not target asequence in the subject.

An iRNA agent can have a first and a second strand chosen from thefollowing:

a first strand which does not target a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which does not target a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a first strand which does not target a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a first strand which does not target a sequence and which has an NRMmodification at the cleavage site or in the cleavage region;

a first strand which does not target a sequence and which has an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end; and

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end(5′ end NRM modifications are preferentially not at the terminus butrather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of anantisense strand);

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a second strand which targets a sequence and which preferably does nothave an an NRM modification at the cleavage site or in the cleavageregion;

a second strand which targets a sequence and which does not have an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end (5′ end NRMmodifications are preferentially not at the terminus but rather at aposition 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisensestrand).

An iRNA agent can also target two sequences and can have a first andsecond strand chosen from:

a first strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end(5′ end NRM modifications are preferentially not at the terminus butrather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of anantisense strand);

a first strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a first strand which targets a sequence and which preferably does nothave an NRM modification at the cleavage site or in the cleavage region;

a first strand which targets a sequence and which dose not have an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end (5′ end NRMmodifications are preferentially not at the terminus but rather at aposition 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisensestrand) and

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end(5′ end NRM modifications are preferentially not at the terminus butrather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of anantisense strand);

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a second strand which targets a sequence and which preferably does nothave an an NRM modification at the cleavage site or in the cleavageregion;

a second strand which targets a sequence and which dose not have an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end (5′ end NRMmodifications are preferentially not at the terminus but rather at aposition 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisensestrand).

Ribose Mimics: The monomers and methods described herein can be used toprepare an RNA, e.g., an iRNA agent, that incorporates a ribose mimic,such as those described herein and those described in copending co-ownedUnited States Provisional Application Ser. No. 60/454,962, filed on Mar.13, 2003, and International Application No. PCT/US04/07070, both ofwhich are hereby incorporated by reference.

Thus, an aspect of the invention features an iRNA agent that includes asecondary hydroxyl group, which can increase efficacy and/or confernuclease resistance to the agent. Nucleases, e.g., cellular nucleases,can hydrolyze nucleic acid phosphodiester bonds, resulting in partial orcomplete degradation of the nucleic acid. The secondary hydroxy groupconfers nuclease resistance to an iRNA agent by rendering the iRNA agentless prone to nuclease degradation relative to an iRNA which lacks themodification. While not wishing to be bound by theory, it is believedthat the presence of a secondary hydroxyl group on the iRNA agent canact as a structural mimic of a 3′ ribose hydroxyl group, thereby causingit to be less susceptible to degradation.

Combination Therapy

In one aspect, composition of the invention can be used in combinationtherapy. The term “combination therapy” includes the administration ofthe subject compounds in further combination with other biologicallyactive ingredients (such as, but not limited to, a second and differentantineoplastic agent) and non-drug therapies (such as, but not limitedto, surgery or radiation treatment). For instance, the compounds of theinvention can be used in combination with other pharmaceutically activecompounds, preferably compounds that are able to enhance the effect ofthe compounds of the invention. The compounds of the invention can beadministered simultaneously (as a single preparation or separatepreparation) or sequentially to the other drug therapy. In general, acombination therapy envisions administration of two or more drugs duringa single cycle or course of therapy.

In one aspect of the invention, the subject compounds may beadministered in combination with one or more separate agents thatmodulate protein kinases involved in various disease states. Examples ofsuch kinases may include, but are not limited to: serine/threoninespecific kinases, receptor tyrosine specific kinases and non-receptortyrosine specific kinases. Serine/threonine kinases include mitogenactivated protein kinases (MAPK), meiosis specific kinase (MEK), RAF andaurora kinase. Examples of receptor kinase families include epidermalgrowth factor receptor (EGFR) (e.g. HER2/neu, HER3, HER4, ErbB, ErbB2,ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor(e.g. FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R);hepatocyte growth/scatter factor receptor (HGFR) (e.g, MET, RON, SEA,SEX); insulin receptor (e.g. IGFI-R); Eph (e.g. CEK5, CEK8, EBK, ECK,EEK, EHK-I, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDK5, SEK); AxI (e.g.Mer/Nyk, Rse); RET; and platelet-derived growth factor receptor (PDGFR)(e.g. PDGFα-R, PDGβ-R, CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1,FLT3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but arenot limited to, BCR-ABL (e.g. p43^(abl), ARG); BTK (e.g. ITK/EMT, TEC);CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.

In another aspect of the invention, the subject compounds may beadministered in combination with one or more agents that modulatenon-kinase biological targets or processes. Such targets include histonedeacetylases (HDAC), DNA methyltransferase (DNMT), heat shock proteins(e.g. HSP90), and proteosomes.

In one embodiment, subject compounds may be combined with antineoplasticagents (e.g. small molecules, monoclonal antibodies, antisense RNA, andfusion proteins) that inhibit one or more biological targets such asZolinza, Tarceva, Iressa, Tykerb, Gleevec, Sutent, Sprycel, Nexavar,Sorafinib, CNF2024, RG108, BMS387032, Affmitak, Avastin, Herceptin,Erbitux, AG24322, PD325901, ZD6474, PD 184322,

Obatodax, ABT737 and AEE788. Such combinations may enhance therapeuticefficacy over efficacy achieved by any of the agents alone and mayprevent or delay the appearance of resistant mutational variants.

In certain preferred embodiments, the compounds of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents encompass a wide range of therapeutic treatmentsin the field of oncology. These agents are administered at variousstages of the disease for the purposes of shrinking tumors, destroyingremaining cancer cells left over after surgery, inducing remission,maintaining remission and/or alleviating symptoms relating to the canceror its treatment. Examples of such agents include, but are not limitedto, alkylating agents such as mustard gas derivatives

(Mechlorethamine, cylophosphamide, chlorambucil, melphalan, ifosfamide),ethylenimines (thiotepa, hexamethylmelanine), Alkylsulfonates(Busulfan), Hydrazines and Triazines (Altretamine, Procarbazine,Dacarbazine and Temozolomide), Nitrosoureas (Carmustine, Lomustine andStreptozocin), Ifosfamide and metal salts (Carboplatin, Cisplatin, andOxaliplatin); plant alkaloids such as Podophyllotoxins (Etoposide andTenisopide), Taxanes (Paclitaxel and Docetaxel), Vinca alkaloids(Vincristine, Vinblastine, Vindesine and Vinorelbine), and Camptothecananalogs (Ironotecan and Topotecan); anti-tumor antibiotics such asChromomycins (Dactinomycin and Plicamycin), Anthracyclines (Doxorubicin,Daunorubicin, Epirubicin, Mitoxantrone, Valrubicin and Idarubicin), andmiscellaneous antibiotics such as Mitomycin, Actinomycin and Bleomycin;anti-metabolites such as folic acid antagonists (Methotrexate,Pemetrexed, Raltitrexed, Aminopterin), pyrimidine antagonists(5-Fluorouracil, Floxuridine, Cytarabine, Capecitabine, andGemcitabine), purine antagonists (6-Mercaptopurine and 6-Thioguanine)and adenosine deaminase inhibitors (Cladribine, Fludarabine,Mercaptopurine, Clofarabine, Thioguanine, Nelarabine and Pentostatin);topoisomerase inhibitors such as topoisomerase I inhibitors (Ironotecan,topotecan) and topoisomerase II inhibitors (Amsacrine, etoposide,etoposide phosphate, teniposide); monoclonal antibodies (Alemtuzumab,Gemtuzumab ozogamicin, Rituximab, Trastuzumab, Ibritumomab Tioxetan,Cetuximab, Panitumumab, Tositumomab, Bevacizumab); and miscellaneousanti-neoplasties such as ribonucleotide reductase inhibitors(Hydroxyurea); adrenocortical steroid inhibitor (Mitotane); enzymes(Asparaginase and Pegaspargase); anti-microtubule agents (Estramustine);and retinoids (Bexarotene, Isotretinoin, Tretinoin (ATRA). In certainpreferred embodiments, the compounds of the invention are administeredin combination with a chemoprotective agent. Chemoprotective agents actto protect the body or minimize the side effects of chemotherapy.Examples of such agents include, but are not limited to, amfostine,mesna, and dexrazoxane.

In one aspect of the invention, the subject compounds are administeredin combination with radiation therapy. Radiation is commonly deliveredinternally (implantation of radioactive material near cancer site) orexternally from a machine that employs photon (x-ray or gamma-ray) orparticle radiation. Where the combination therapy further comprisesradiation treatment, the radiation treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and radiation treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the radiation treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

It will be appreciated that compounds of the invention can be used incombination with an immunotherapeutic agent. One form of immunotherapyis the generation of an active systemic tumor-specific immune responseof host origin by administering a vaccine composition at a site distantfrom the tumor. Various types of vaccines have been proposed, includingisolated tumor-antigen vaccines and anti-idiotype vaccines. Anotherapproach is to use tumor cells from the subject to be treated, or aderivative of such cells (reviewed by Schirrmacher et al. (1995) J.Cancer Res. Clin. Oncol. 121:487). In U.S. Pat. No. 5,484,596, Hanna Jr.et al. claim a method for treating a resectable carcinoma to preventrecurrence or metastases, comprising surgically removing the tumor,dispersing the cells with collagenase, irradiating the cells, andvaccinating the patient with at least three consecutive doses of about10⁷ cells.

It will be appreciated that the compounds of the invention mayadvantageously be used in conjunction with one or more adjunctivetherapeutic agents. Examples of suitable agents for adjunctive therapyinclude steroids, such as corticosteroids (amcinonide, betamethasone,betamethasone dipropionate, betamethasone valerate, budesonide,clobetasol, clobetasol acetate, clobetasol butyrate, clobetasol17-propionate, cortisone, deflazacort, desoximetasone, diflucortolonevalerate, dexamethasone, dexamethasone sodium phosphate, desonide,furoate, fluocinonide, fluocinolone acetonide, halcinonide,hydrocortisone, hydrocortisone butyrate, hydrocortisone sodiumsuccinate, hydrocortisone valerate, methyl prednisolone, mometasone,prednicarbate, prednisolone, triamcinolone, triamcinolone acetonide, andhalobetasol proprionate); a 5HTi agonist, such as a triptan (e.g.sumatriptan or naratriptan); an adenosine A1 agonist; an EP ligand; anNMDA modulator, such as a glycine antagonist; a sodium channel blocker(e.g. lamotrigine); a substance P antagonist (e.g. an NKi antagonist); acannabinoid; acetaminophen or phenacetin; a 5-lipoxygenase inhibitor; aleukotriene receptor antagonist; a DMARD (e.g. methotrexate); gabapentinand related compounds; a tricyclic antidepressant (e.g. amitryptilline);a neurone stabilising antiepileptic drug; a mono-aminergic uptakeinhibitor (e.g. venlafaxine); a matrix metalloproteinase inhibitor; anitric oxide synthase (NOS) inhibitor, such as an iNOS or an nNOSinhibitor; an inhibitor of the release, or action, of tumour necrosisfactor α; an antibody therapy, such as a monoclonal antibody therapy; anantiviral agent, such as a nucleoside inhibitor (e.g. lamivudine) or animmune system modulator (e.g. interferon); an opioid analgesic; a localanaesthetic; a stimulant, including caffeine; an H2-antagonist (e.g.ranitidine); a proton pump inhibitor (e.g. omeprazole); an antacid (e.g.aluminium or magnesium hydroxide; an antiflatulent (e.g. simethicone); adecongestant (e.g. phenylephrine, phenylpropanolamine, pseudoephedrine,oxymetazoline, epinephrine, naphazoline, xylometazoline,propylhexedrine, or levo-desoxyephedrine); an antitussive (e.g. codeine,hydrocodone, carmiphen, carbetapentane, or dextramethorphan); adiuretic; or a sedating or non-sedating antihistamine.

Pharmaceutical compositions. In one embodiment, the invention relates toa pharmaceutical composition containing an iRNA agent of the presentinvention, as described in the preceding sections, and apharmaceutically acceptable carrier, as described below. Apharmaceutical composition including the modified iRNA agent is usefulfor treating a disease caused by expression of a target gene. In thisaspect of the invention, the iRNA agent of the invention is formulatedas described below. The pharmaceutical composition is administered in adosage sufficient to inhibit expression of the target gene.

The pharmaceutical compositions of the present invention areadministered in dosages sufficient to inhibit the expression or activityof the target gene. Compositions containing the iRNA agent of theinvention can be administered at surprisingly low dosages. A maximumdosage of 5 mg iRNA agent per kilogram body weight per day may besufficient to inhibit or completely suppress the expression or activityof the target gene.

In general, a suitable dose of modified iRNA agent will be in the rangeof 0.001 to 500 milligrams per kilogram body weight of the recipient perday (e.g., about 1 microgram per kilogram to about 500 milligrams perkilogram, about 100 micrograms per kilogram to about 100 milligrams perkilogram, about 1 milligrams per kilogram to about 75 milligrams perkilogram, about 10 micrograms per kilogram to about 50 milligrams perkilogram, or about 1 microgram per kilogram to about 50 micrograms perkilogram). The pharmaceutical composition may be administered once perday, or the iRNA agent may be administered as two, three, four, five,six or more sub-doses at appropriate intervals throughout the day. Inthat case, the iRNA agent contained in each sub-dose must becorrespondingly smaller in order to achieve the total daily dosage. Thedosage unit can also be compounded for delivery over several days, e.g.,using a conventional sustained release formulation which providessustained release of the iRNA agent over a several day period. Sustainedrelease formulations are well known in the art. In this embodiment, thedosage unit contains a corresponding multiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the infection or disease, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNA agent encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases. For example, mouse repositories canbe found at The Jackson Laboratory, Charles River Laboratories, Taconic,Harlan, Mutant Mouse Regional Resource Centers (MMRRC) National Networkand at the European Mouse Mutant Archive. Such models may be used for invivo testing of iRNA agent, as well as for determining a therapeuticallyeffective dose.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), ocular,rectal, vaginal and topical (including buccal and sublingual)administration. In preferred embodiments, the pharmaceuticalcompositions are administered by intravenous or intraparenteral infusionor injection. The pharmaceutical compositions can also be administeredintraparenchymally, intrathecally, and/or by stereotactic injection.

For oral administration, the iRNA agent useful in the invention willgenerally be provided in the form of tablets or capsules, as a powder orgranules, or as an aqueous solution or suspension.

Tablets for oral use may include the active ingredients mixed withpharmaceutically acceptable excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavoring agents, coloring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid or talc. If desired, the tablets may be coatedwith a material such as glyceryl monostearate or glyceryl distearate, todelay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the activeingredient is mixed with a solid diluent, and soft gelatin capsuleswherein the active ingredient is mixed with water or an oil such aspeanut oil, liquid paraffin or olive oil.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,the pharmaceutical compositions of the invention will generally beprovided in sterile aqueous solutions or suspensions, buffered to anappropriate pH and isotonicity. Suitable aqueous vehicles includeRinger's solution and isotonic sodium chloride. In a preferredembodiment, the carrier consists exclusively of an aqueous buffer. Inthis context, “exclusively” means no auxiliary agents or encapsulatingsubstances are present which might affect or mediate uptake of iRNAagent in the cells that harbor the target gene or virus. Such substancesinclude, for example, micellar structures, such as liposomes or capsids,as described below. Although microinjection, lipofection, viruses,viroids, capsids, capsoids, or other auxiliary agents are required tointroduce iRNA agent into cell cultures, surprisingly these methods andagents are not necessary for uptake of iRNA agent in vivo. The iRNAagent of the present invention are particularly advantageous in thatthey do not require the use of an auxiliary agent to mediate uptake ofthe iRNA agent into the cell, many of which agents are toxic orassociated with deleterious side effects. Aqueous suspensions accordingto the invention may include suspending agents such as cellulosederivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth,and a wetting agent such as lecithin. Suitable preservatives for aqueoussuspensions include ethyl and n-propyl p-hydroxybenzoate.

The pharmaceutical compositions can also include encapsulatedformulations to protect the iRNA agent against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, bio compatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811; PCT publication WO 91/06309; and European patent publicationEP-A-43075, which are incorporated by reference herein.

Toxicity and therapeutic efficacy of iRNA agent can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.iRNA agents that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosages ofcompositions of the invention are preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anyiRNA agent used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the iRNA agent or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test iRNA agent which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In addition to their administration individually or as a plurality, asdiscussed above, iRNA agents relating to the invention can beadministered in combination with other known agents effective intreating viral infections and diseases. In any event, the administeringphysician can adjust the amount and timing of iRNA agent administrationon the basis of results observed using standard measures of efficacyknown in the art or described herein.

For oral administration, the iRNA agent useful in the invention willgenerally be provided in the form of tablets or capsules, as a powder orgranules, or as an aqueous solution or suspension.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limitedto, oral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), ocular,rectal, vaginal, and topical (including buccal and sublingual)administration. In preferred embodiments, the pharmaceuticalcompositions are administered by intravenous or intraparenteral infusionor injection. The pharmaceutical compositions can also be administeredintraparenchymally, intrathecally, and/or by stereotactic injection.

Methods for identifying iRNA agents having increased stability. In yetanother aspect, the invention relates to methods for identifying iRNAagent having increased stability in biological tissues and fluids suchas serum. iRNA agent having increased stability have enhanced resistanceto degradation, e.g., by chemicals or nucleases (particularlyendonucleases) which normally degrade RNA molecules. Methods fordetecting increases in nucleic acid stability are well known in the art.Any assay capable of measuring or detecting differences between a testiRNA agent and a control iRNA agent in any measurable physical parametermay be suitable for use in the methods of the present invention. Ingeneral, because the inhibitory effect of an iRNA agent on a target geneactivity or expression requires that the molecule remain intact, thestability of a particular iRNA agent can be evaluated indirectly byobserving or measuring a property associated with the expression of thegene. Thus, the relative stability of an iRNA agent can be determined byobserving or detecting (1) an absence or observable decrease in thelevel of the protein encoded by the target gene, (2) an absence orobservable decrease in the level of mRNA product from the target gene,and (3) a change or loss in phenotype associated with expression of thetarget gene. In the context of a medical treatment, the stability of aniRNA agent may be evaluated based on the degree of the inhibition ofexpression or function of the target gene, which in turn may be assessedbased on a change in the disease condition of the patient, such asreduction in symptoms, remission, or a change in disease state.

In one embodiment, the method includes preparing an iRNA agent asdescribed above (e.g., through chemical synthesis), incubating the iRNAagent with a biological sample, then analyzing and identifying thoseiRNA agent that exhibit an increased stability as compared to a controliRNA agent.

In an exemplified embodiment, iRNA agent is produced in vitro bymixing/annealing complementary single-stranded RNA strands, preferablyin a molar ratio of at least about 3:7, more preferably in a molar ratioof about 4:6, and most preferably in essentially equal molar amounts(e.g., a molar ratio of about 5:5). Preferably, the single-stranded RNAstrands are denatured prior to mixing/annealing, and the buffer in whichthe mixing/annealing reaction takes place contains a salt, preferablypotassium chloride. Single-stranded RNA strands may be synthesized bysolid phase synthesis using, for example, an Expedite 8909 synthesizer(Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany), asdescribed above.

iRNA agent are incubated with a biological sample under the conditionssufficient or optimal for enzymatic function. After incubating with abiological sample, the stability of the iRNA agent is analyzed by meansconventional in the art, for example using RNA gel electrophoresis asexemplified herein. For example, when the sample is serum, the iRNAagent may be incubated at a concentration of 1-10 μM, preferably 2-8 μM,more preferably 3-6 μM, and most preferably 4-5 μM. The incubationtemperature is preferably between 25° C. and 45° C., more preferablybetween 35° C. and 40° C., and most preferably about 37° C.

The biological sample used in the incubation step may be derived fromtissues, cells, biological fluids or isolates thereof. For example, thebiological sample may be isolated from a subject, such as a wholeorganism or a subset of its tissues or cells. The biological sample mayalso be a component part of the subject, such as a body fluid, includingbut not limited to blood, serum, plasma, mucus, lymphatic fluid,synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amnioticcord blood, urine, vaginal fluid and semen. Preferably, the biologicalsample is a serum derived from a blood sample of a subject. The subjectis preferably a mammal, more preferably a human or a mouse.

In another embodiment, the method includes selecting an iRNA agenthaving increased stability by measuring the mRNA and/or proteinexpression levels of a target gene in a cell following introduction ofthe iRNA agent. In this embodiment, an iRNA agent of the inventioninhibits expression of a target gene in a cell, and thus the methodincludes selecting an iRNA agent that induces a measurable reduction inexpression of a target gene as compared to a control iRNA agent. Assaysthat measure gene expression by monitoring RNA and/or protein levels canbe performed within about 24 hours following uptake of the iRNA agent bythe cell. For example, RNA levels can be measured by Northern blottechniques, RNAse Protection Assays, or Quality Control-PCR (QC-PCR)(including quantitative reverse transcription coupled PCR(RT-PCR)) andanalogous methods known in the art. Protein levels can be assayed, forexample, by Western blot techniques, flow cytometry, or reporter geneexpression (e.g., expression of a fluorescent reporter protein, such asgreen fluorescent protein (GFP)). RNA and/or protein levels resultingfrom target gene expression can be measured at regular time intervalsfollowing introduction of the test iRNA agent, and the levels arecompared to those following introduction of a control iRNA agent intocells. A control iRNA agent can be a nonsensical iRNA agent (i.e., aniRNA agent having a scrambled sequence that does not target anynucleotide sequence in the subject), an iRNA agent that can target agene not present in the subject (e.g., a luciferase gene, when the iRNAagent is tested in human cells), or an iRNA agent otherwise previouslyshown to be ineffective at silencing the target gene. The mRNA andprotein levels of the test sample and the control sample can becompared. The test iRNA agent is selected as having increased stabilitywhen there is a measurable reduction in expression levels followingabsorption of the test iRNA agent as compared to the control iRNA agent.mRNA and protein measurements can be made using any art-recognizedtechnique (see, e.g., Chiang, M. Y., et al., J. Biol. Chem. (1991)266:18162-71; Fisher, T, et al., Nucl. Acids Res. (1993) 21:3857; andChen et al., J. Biol. Chem. (1996) 271:28259).

Methods for identifying iRNA agents with ability to inhibit geneexpression. The ability of an iRNA agent composition of the invention toinhibit gene expression can be measured using a variety of techniquesknown in the art. For example, Northern blot analysis can be used tomeasure the presence of RNA encoding a target protein. The level of thespecific mRNA produced by the target gene can be measured, e.g., usingRT-PCR. Because iRNA agent directs the sequence-specific degradation ofendogenous mRNA through RNAi, the selection methods of the inventionencompass any technique that is capable of detecting a measurablereduction in the target RNA. In yet another example, Western blots canbe used to measure the amount of target protein present. In stillanother embodiment, a phenotype influenced by the amount of the proteincan be detected. Techniques for performing Western blots are well knownin the art (see, e.g., Chen, et al., J. Biol. Chem. (1996) 271:28259).

When the target gene is to be silenced by an iRNA agent that targets apromoter sequence of the target gene, the target gene can be fused to areporter gene, and reporter gene expression (e.g., transcription and/ortranslation) can be monitored. Similarly, when the target gene is to besilenced by an iRNA agent that targets a sequence other than a promoter,a portion of the target gene (e.g., a portion including the targetsequence) can be fused with a reporter gene so that the reporter gene istranscribed. By monitoring a change in the expression of the reportergene in the presence of the iRNA agent, it is possible to determine theeffectiveness of the iRNA agent in inhibiting the expression of thereporter gene. The expression levels of the reporter gene in thepresence of the test iRNA agent versus a control iRNA agent are thencompared. The test iRNA agent is selected as having increased stabilitywhen there is a measurable reduction in expression levels of thereporter gene as compared to the control iRNA agent. Examples ofreporter genes useful for use in the present invention include, withoutlimitation, those coding for luciferase, GFP, chloramphenicol acetyltransferase (CAT), β-galactosidase, and alkaline phosphatase. Suitablereporter genes are described, for example, in Current Protocols inMolecular Biology, John Wiley & Sons, New York (Ausubel, F. A., et al.,eds., 1989); Gould, S. J., and S. Subramani, Anal. Biochem. (1988)7:404-408; Gorman, C. M., et al., Mol. Cell. Biol. (1982) 2:1044-1051;and Selden, R., et al., Mol. Cell. Biol. (1986) 6:3173-3179; each ofwhich is hereby incorporated by reference.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Synthetic Methods

The iRNA agents of the present invention can be prepared according tostandard procedures known in the art such as in the following referenceswhich are incorporated by the reference by its entirety:

-   1. Chemical synthesis of oligonucleotide triphosphates.    Vaghefi, M. M. Editor(s): Vaghefi, Morteza. Nucleoside Triphosphates    and Their Analogs, 2005, 105-113.-   2. Solid phase synthesis of 5′-diphosphorylated oligoribonucleotides    and their conversion to capped m7 Gppp-oligoribonucleotides for use    as primers for influenza A virus RNA polymerase in vitro.    Brownlee, G. G.; Fodor, E.; Pritlove, D.C.; Gould, K. G.;    Dalluge, J. J., Nucleic Acids Res. 1995, 23(14), 2641-7.-   3. Chemical synthesis of 5′-pyrophosphate and triphosphate    derivatives of 3′,5′ ApA, ApG, GpA and GpG. CD study of the effect    of 5′-phosphate groups on the conformation of 3′,5′ GpG. Tomasz, J.;    Simoncsits, A.; Kajtar, M.; Krug, R. M.; Shatkin, A. J., Nucleic    Acids Res., 1978, 5(8), 2945-57.-   4. Chemical synthesis of an effective inhibitor or protein synthesis    in euharytic cells: pppA2′, 5′A2′, 5′A and some analogues.    Hartog, J. A. J. den; Wijnands, R. A.; van Boom, J. H.; Nucleic    Acids Symp. Series No 7, 1980, 157-166.-   5. Rapid and efficient synthesis of nucleoside    5′-0-(1-thiotriphosphates), 5′-triphosphates and    2′,3′-cyclophosphorothioates using    2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one. Ludwig, Janos;    Eckstein, Fritz., J. Org. Chem., 1989, 54(3), 631-35.-   6. Novel solid phase synthesis of 2′-O-methylribonucleoside    5′-triphosphates and their α-thio analogs. Gaur, R. K.; Sproat, B.    S.; Krupp, Guido, Tetrahedron Lett. 1992, 33(23), 3301-4.-   7. Preparation of oligodeoxynucleotide 5′-triphosphates using solid    support approach. Lebedev, A. V.; Koukhareva, I. I.; Beck, T;.    Vaghefi, M. M. Nucleosides, Nucleotides & Nucleic Acids, 2001,    20(4-7),1403-1409.-   8. Synthesis and properties of NTP analogs with modified    triphosphate chains. Koukhareva, Inna; Vaghefi, Morteza; Lebedev,    Alexandre. Editor(s): Vaghefi, M. Nucleoside Triphosphates and Their    Analogs, 2005, 39-104 (and references cited there in)-   9. Nucleoside Triphosphates and their Analogs: Chemistry,    Biotechnology, and Biological Applications. Vaghefi, M.; Editor.    USA. 2005, 393 pp. Publisher: CRC Press LLC, Boca Raton, Fla. (and    references cited there in).

EXAMPLES RNA 5′ Triphosphate Synthesis

Syntheses of RNA 5′ triphosphate involving various 2′ O protecting groupstrategies as well as various chemistries for the introduction of the 5′triphosphate moiety or modified analog.

RNA 5′-triphosphates (RNATP) can be efficiently prepared in variousmanners involving alternate protecting groups for the 2′ hydroxyls. Allthe 2′ protecting groups developed and reported so far, (Reviewed byBeaucage, Cur. Op. Drug Disc. Dev., 2008, 11, 203) can be used for thesynthesis of RNATP. These include and are not limited to the well knownfluoro-labile groups such as TBDMS, TOM, CEM and others, but protectinggroups that are cleaved in non fluoride ion conditions can also be ofparticular interest as well. This is because extended fluoride iontreatment and/or heating procedures can damage the RNATP and diminishyields. Likewise, extended purification/desalting procedures will havethe same effect.

All these alternate syntheses involve the use of readily available 3′phosphoramidite building blocks (compounds a in the schemes) as monomerbuilding blocks for automated RNA synthesis. The choice of protectinggroups for the exocyclic amino groups of the nucleobases of thesemonomer building blocks (noted B^(PG) in the schemes) can also bemodulated as base labile (fast labile) protecting groups arepreferentially used (i.e. acetyl for C, phenoxyacetyl (Pac) for A and Gand/or tert-butylphenoxyacetyl (tBuPac) and iso-propylphenoxyacetyl(iPrPac) for G. These fast labile protecting groups have the advantageto be removed without heating the reaction mixture, as it is needed forstandard protecting groups. This later point improves the yields oftarget RNATP.

Examples of RNATP synthesis, presenting various 2′ protecting groups,all using the Ludwig phosphitylation reagent (Ludwig and Ekcstein, 1989,J. Org. Chem., 54, 631), are described.

Scheme 6: Target RNATP can be synthesized using the 2′tert-butyldithiomethyl (DTM) protecting group (Kwiatkowski et al., 2006,J. Am. Chem. Soc., 128, 12356). The corresponding nucleobase protected(standard or fast labile PG) 5′ DMTr 3′ phosphoramidites (101a) are usedin the automated RNA synthesis using the standard conditions on solidsupport (succinyl linked LCAA CPG). Other supports and linkers can bereadily used as well. The resulting solid supported oligonucleotide,lacking the 5′ terminal DMTr group (5′ OH, compound 101b) is involved ina phosphitylation reaction using excess salicylophosphorochloridite inpyridine (Ludwig and Ekcstein, 1989, J. Org. Chem., 54, 631). Aftershaking the solid support with the 0.5 M solution of phosphitylationreagent at room temperature for 30 min, the solution is washed off andthe solid support is well rinsed with dry acetonitrile. 0.5 M solutionof tributylammonium pyrophosphate in DMF is then introduced to the solidsupported oligonucleotide and shaken for 24 h. After washing off thesolution, the resulting oligonucleotide (101c) is oxidized or sulfurizedusing standard reagents. The corresponding cyclic TP (101d) is thenhydrolyzed upon treatment with ammonia, allowing at the same timedeblocking of the oligonucleotide from the solid support and nucleobasedeprotection. Finally, removal of all the 2′ DTM groups using a reducingagent such as DTT or TCEP in a pH 7.6 buffer at 55° C. provides thetarget RNATP or RNA(αS)TP.

Scheme 7: Target RNATP can be synthesized using the 2′ acetal levulinyl(ALE) protecting group (Damha et al, 2009, J. Am. Chem. Soc, 131, 8496).The corresponding base protected (PG=levulinyl for A and C, DMF for G),5′ DMTr 3′ phosphoramidites (102a) are used in the automated RNAsynthesis using the standard conditions on solid support (Q linker LCAACPG is used with this 2′ protecting group). Other supports and linkerscan be possibly used as well. The resulting solid supportedoligonucleotide, lacking the 5′ terminal DMTr group (5′ OH, compound102b) is involved in a phosphitylation reaction using excesssalicylophosphorochloridite in pyridine. After shaking the solid supportwith the phosphitylation reagent at room temperature for 30 min, thesolution is washed off and the solid support is well rinsed with dryacetonitrile. 0.5 M solution of tributylammonium pyrophosphate in DMF isthen introduced to the solid supported oligonucleotide and shaken for 24h. After washing off the solution, the resulting oligonucleotide (102c)is oxidized or sulfurized using standard reagents. The correspondingcyclic TP (102d) is then hydrolyzed by adding water in ACN, then rinsedwith dry ACN and well dried under Argon flush. Particular deblockingconditions are used for the ALE group, i.e. firstly the backbonecyanoethyl groups are removed upon treatment with triethylamine inanhydrous acetonitrile. Then the 2′ ALE groups, together with the baseprotecting groups are cleaved upon treatment with hydrazine hydrate in amixture buffer of AcOH and pyridine. Finally cleavage of the Q linkerwith 1 M TBAF at room temperature provides the target RNATP orRNA(αS)TP.

Scheme 8: Target RNATP can be synthesized using the 2′ bisacetoxyethoxymethyl (ACE) protecting group (Caruthers et al., 1998, J.Am. Chem. Soc., 120, 11820). The corresponding base protected (standardor fast labile PG) 5′ silyl (DOD) 3′ phosphoramidites (103a) arecommercially available and are used in the automated RNA synthesis usingthe standard conditions on solid support (succinyl linked LCAA CPG).Other supports and linkers can be readily used as well. The resultingsolid supported oligonucleotide, lacking the 5′ terminal silyl group (5′OH, compound 103b) is involved in a phosphitylation reaction usingexcess salicylophosphorochloridite in pyridine. After shaking the solidsupport with the 0.5 M solution of phosphitylation reagent at roomtemperature for 30 min, the solution is washed off and the solid supportis well rinsed with dry acetonitrile. 0.5 M solution of tributylammoniumpyrophosphate in DMF is then introduced to the solid supportedoligonucleotide and shaken for 24 h. After washing off the solution, theresulting oligonucleotide (103c) is oxidized or sulfurized usingstandard reagents. The corresponding cyclic TP (103d) is then hydrolyzedupon treatment with ammonia, allowing at the same time deblocking of theoligonucleotide from the solid support and nucleobase deprotection.Finally, removal of all the 2′ ACE groups using a slightly acidic pH 3buffer provides the target RNATP or RNA(αS)TP.

Scheme 9: Target RNATP can be synthesized using the acid labile 2′ Ctmpor Fpmp protecting group (Reese et al., 1994, Nucleic Acids Res., 22,2209). The corresponding base protected (standard or fast labile PG) 5′DMTr 3′ phosphoramidites (104a) are commercially available and used inthe automated RNA synthesis using the standard conditions on solidsupport (succinyl linked LCAA CPG). Other supports and linkers can bereadily used as well. The resulting solid supported oligonucleotide,lacking the 5′ terminal DMTr group (5′ OH, compound 104b) is involved ina phosphitylation reaction using excess salicylophosphorochloridite inpyridine. After shaking the solid support with the 0.5 M solution ofphosphitylation reagent at room temperature for 30 min, the solution iswashed off and the solid support is well rinsed with dry acetonitrile.0.5 M solution of tributylammonium pyrophosphate in DMF is thenintroduced to the solid supported oligonucleotide and shaken for 24 h.After washing off the solution, the resulting oligonucleotide (104c) isoxidized or sulfurized using standard reagents. The corresponding cyclicTP (104d) is then hydrolyzed upon treatment with ammonia, allowing atthe same time deblocking of the oligonucleotide from the solid supportand nucleobase deprotection. Finally, removal of all the 2′ Ctmp/Ftmpgroups using an acidic pH 2.0-2.3 buffer provides the target RNATP orRNA(αS)TP.

In combination with the variation of 2′ protecting groups and/ornucleobase standard or fast labile protecting groups, several differentways, other than the Ludwig-Eckstein method, previously depicted, ofintroducing the triphosphate (or modified α-thio or α-borano analog)moiety to the solid supported oligonucleotide, can be applied. Theseinclude the use and the preparation of various 5′ bis(mono orpolysubstituted) aryl phosphite triesters, 5′ phosphorodiamidites, 5′oxo thia or dithiaphospho-2-lanes or solid supported 5′mono(thio)phosphates. Some examples are described hereafter. For thesynthesis of RNA 5′ alpha thio triphosphates and 5′ alpha dithiotriphosphates, the use and preparation of 5′ H-phosphonothioates and 5′H-phosphonodithioates, using diphenyl thiophosphite is described.

Schemes 5-8: In a general way, the salicyl part of the Ludwig reagentcan be replaced by a bis aryl moiety, which is mono or polysubstitutedby different electron withdrawal groups (EWG). Such EWG can be nitro,cyano, ortho-chloro, ortho-fluoro, ortho,para di-chloro,ortho,para-di-flluoro, pentachloro, pentafluoro and others. The use ofsuch O,O-bis aryl phosphorochloridites affords, after reaction withtributylammonium pyrophosphate, the cyclic intermediate (c). After theoxidizing/sulfurization or borane complexation step, followed bydeprotection and support cleavage steps, the target RNATP or modifiedanalog is obtained in similar fashion. Some examples exhibiting thevarious 2′ protecting groups are depicted in schemes 5-8 and will bebriefly described hereafter.

Scheme 10: The most commonly used 2′ TBDMS protected 5′ DMTr 3′phosphoramidites (105a) and standard solid supported RNA synthesisconditions can be used for the construction of the 5′ OH oligonucleotide(compound 105b). It is then involved in a phosphitylation reaction usingexcess O,O-bis aryl phosphorochloridites in solution in dry pyridine.After shaking the solid support with the 0.5 M solution ofphosphitylation reagent at room temperature for 30 min, the solution iswashed off and the solid support is well rinsed with dry acetonitrileand dried with Argon flux. 0.5 M solution of tributylammoniumpyrophosphate in DMF is then introduced to the solid supportedoligonucleotide and shaken for 24 h. After washing off the solution, theresulting oligonucleotide (105c) is oxidized, sulfurized or reacted witha suitable boron complex, using standard reagents. The correspondingcyclic TP (105d) is then hydrolyzed upon treatment with ammonia,allowing at the same time deblocking of the oligonucleotide from thesolid support and nucleobase deprotection. Finally, removal of all the2′ TBDMS groups using HF-pyridine provides the target RNATP, RNA(αS)TPor RNA(αBH₃)TP.

Scheme 11: The 2′ ALE protected 5′ DMTr 3′ phosphoramidites (102a) andstandard solid supported RNA synthesis conditions can be used for theconstruction of the 5′ OH oligonucleotide (compound 102b), as previouslydiscussed for scheme 2. It is then involved in a phosphitylationreaction using excess O,O,O-tris 4-nitrophenyl phosphite in solution indry pyridine. After shaking the solid support with the 0.5 M solution ofphosphitylation reagent at room temperature for 30 min, the solution iswashed off and the solid support is well rinsed with dry acetonitrile.0.5 M solution of tributylammonium pyrophosphate in DMF is thenintroduced to the solid supported oligonucleotide and shaken for 24 h.After washing off the solution, the resulting oligonucleotide (102c) isoxidized, sulfurized or reacted with a suitable boron complex, usingstandard reagents. Previously described hydrolysis and deblocking stepsof (102d) afford the target RNATP, RNA(αS)TP or RNA(αBH₃)TP.

Scheme 12: The 2′ ACE protected phosphoramidites (103a) and standardsolid supported RNA synthesis conditions can be used for theconstruction of the 5′ OH oligonucleotide (compound 103b), as previouslydiscussed for scheme 3. It is then involved in a phosphitylationreaction using excess O,O-bis 4-cyano phosphorochloridite in solution indry pyridine. After shaking the solid support with the 0.5 M solution ofphosphitylation reagent at room temperature for 30 min, the solution iswashed off and the solid support is well rinsed with dry acetonitrile.0.5 M solution of tributylammonium pyrophosphate in DMF is thenintroduced to the solid supported oligonucleotide and shaken for 24 h.After washing off the solution, the resulting oligonucleotide (103c) isoxidized, sulfurized or reacted with a suitable boron complex, usingstandard reagents. Previously described hydrolysis and deblocking stepsof (103d) afford the target RNATP, RNA(αS)TP or RNA(αBH₃)TP.

Scheme 13: The 2′ Ctmp/Ftmp protected phosphoramidites (104a) andstandard solid supported RNA synthesis conditions can be used for theconstruction of the 5′ OH oligonucleotide (compound 104b), as previouslydiscussed for scheme 4. It is then involved in a phosphitylationreaction using excess O,O-bis 2,4-dichloro phosphorochloridite insolution in dry pyridine. After shaking the solid support with the 0.5 Msolution of phosphitylation reagent at room temperature for 30 min, thesolution is washed off and the solid support is well rinsed with dryacetonitrile. 0.5 M solution of tributylammonium pyrophosphate in DMF isthen introduced to the solid supported oligonucleotide and shaken for 24h. After washing off the solution, the resulting oligonucleotide (104c)is oxidized, sulfurized or reacted with a suitable boron complex, usingstandard reagents. Previously described hydrolysis and deblocking stepsof (104d) afford the target RNATP, RNA(αS)TP or RNA(αBH₃)TP.

Schemes 14-17: In a general way, oligonucleotide 5′ phosphorodiamiditescan react, in the presence of tetrazole, with tributylammoniumpyrophosphate, affording the previously described cyclic intermediate(c), (Shaw et al., 2003, Org. Lett., 5, 2401; Fischer et al. 2002, J.Med. Chem., 45, 5384). After the oxidizing/sulfurization or boranecomplexation step, followed by deprotection and support cleavage steps,the target RNATP or modified analog is obtained in similar fashion. Someexamples exhibiting the various 2′ protecting groups are depicted inschemes 9-12 and will be briefly described hereafter.

Scheme 14: The 2′ TBDMS protected 5′ DMTr 3′ phosphoramidites (105a) andstandard solid supported RNA synthesis conditions can be used for theconstruction of the 5′ OH oligonucleotide (compound 105b), as previouslydiscussed for scheme 5. It is then involved in a phosphitylationreaction using excess chloro-N,N-tetraisopropyl-phosphoro bis amidite insolution in dry pyridine. After shaking the solid support with the 0.5 Msolution of phosphitylation reagent at room temperature for 30 min, thesolution is washed off and the solid support is well rinsed with dryacetonitrile. 0.5 M solution of tributylammonium pyrophosphate in DMF isthen introduced to the solid supported oligonucleotide and shaken for 24h. After washing off the solution, the resulting oligonucleotide (105c)is oxidized, sulfurized or reacted with a suitable boron complex, usingstandard reagents. The corresponding cyclic TP (105d) is then hydrolyzedand deblocked as previously described, affording the target RNATP,RNA(αS)TP or RNA(αBH₃)TP

Scheme 15: The 2′ Ctmp/Ftmp protected phosphoramidites (104a) andstandard solid supported RNA synthesis conditions can be used for theconstruction of the 5′ OH oligonucleotide (compound 104b), as previouslydiscussed for scheme 4. It is then involved in a phosphitylationreaction using excess N,N,N′,N′,N″,N″-hexaaisopropyl-phosphoro trisamidite in solution of dry ACN containing tetrazole solution. Aftershaking the solid support with the 0.5 M solution of phosphitylationreagent and tetrazole at room temperature for 30 min, the solution iswashed off and the solid support is well rinsed with dry acetonitrile.0.5 M solution of tributylammonium pyrophosphate in DMF is thenintroduced to the solid supported oligonucleotide and shaken for 24 h.After washing off the solution, the resulting oligonucleotide (104c) isoxidized, sulfurized or reacted with a suitable boron complex, usingstandard reagents. Previously described hydrolysis and deblocking stepsof (104d) afford the target RNATP, RNA(αS)TP or RNA(αBH₃)TP.

Scheme 16: The 2′ ACE protected phosphoramidites (103a) and standardsolid supported RNA synthesis conditions can be used for theconstruction of the 5′ OH oligonucleotide (compound 103b), as previouslydiscussed for scheme 3. It is then involved in a phosphitylationreaction using excess chloro-N,N,N′,N′-tetraisopropyl-phosphoro bisamidite in solution in dry pyridine. After shaking the solid supportwith the 0.5 M solution of phosphitylation reagent at room temperaturefor 30 min, the solution is washed off and the solid support is wellrinsed with dry acetonitrile. 0.5 M solution of tributylammoniumpyrophosphate in DMF is then introduced to the solid supportedoligonucleotide and shaken for 24 h. After washing off the solution, theresulting oligonucleotide (103c) is oxidized, sulfurized or reacted witha suitable boron complex, using standard reagents. Previously describedhydrolysis and deblocking steps of (103d) affords the target RNATP,RNA(αS)TP or RNA(αBH₃)TP.

Scheme 17: The 2′ ACE protected phosphoramidites (103a) and standardsolid supported RNA synthesis conditions can be used for theconstruction of the 5′ OH oligonucleotide (compound 103b), as previouslydiscussed for scheme 3. It is then involved in a phosphitylationreaction using excess chloro-phosphoro bis-pyrolo-amidite in solution indry pyridine. Previously described steps for scheme 11 afford the targetRNATP, RNA(αS)TP or RNA(αBH₃)TP.

Schemes 17-19: Oligonucleotide 5′ (2-thio, 2-oxo or 2-borano) oxothia ordithiaphospho-2-lanes can react with inorganic pyrophosphate in thepresence of strong base catalyst DBU to give the corresponding RNATP,RNA(αS)TP, RNA(αS₂)TP, RNA(αBH₃)TP or RNA(αS,BH₃)TP (Okruszek et al.,1994, J. Med. Chem., 37, 3850). This strategy of introduction of themodified TP moiety can be used with variation of the 2′ protectinggroups in a similar manner as previously described. Some examples aredescribed hereafter.

Scheme 18: The 2′ ACE protected phosphoramidites (103a) and standardsolid supported RNA synthesis conditions can be used for theconstruction of the 5′ OH oligonucleotide (compound 103b), as previouslydiscussed for scheme 3. It is then involved in a phosphitylationreaction using excess chloro-phosphoro bis-pyrolo-amidite in solution indry pyridine. Previously described steps for scheme 11 afford the targetRNATP, RNA(αS)TP or RNA(αBH₃)TP.

Scheme 19: The 2′ ACE protected 5′ DMTr 3′ phosphoramidites (103a) andstandard solid supported RNA synthesis conditions are used for theconstruction of the 5′ OH oligonucleotide (compound 103b), as previouslydiscussed for scheme 5. It is then involved in a phosphitylationreaction using excess di-isopropyl oxothiaphospho-2-lane amidite insolution in dry acetonitrile. After shaking the solid support with the0.5 M solution of phosphitylation reagent, premixed with 0.25 M solutionof tetrazole in dry ACN at room temperature for 30 min, the solution iswashed off and the solid support is well rinsed with dry acetonitrile,affording the oligonucleotide 5′ oxothiaphospholane intermediate (108c),which is further oxidized, sulfurized or reacted with a suitable boroncomplex, using standard reagents. 0.5 M solution of tributylammoniumpyrophosphate in DMF is then introduced to the solid supportedoligonucleotide, along with catalytic amount of DBU and shaken for 24 h,providing opening of the dithiaphospholane cycle and elimination of thepending thioethyl group, affording the alpha thio/dithio/thioboranotriphosphate intermediate (109d). This oligonucleotide TP is thenhydrolyzed and deblocked as previously described for scheme 3, affordingthe target RNATP, RNA(αS)TP, or RNA(αBH₃)TP.

Scheme 20: Nucleoside 5′ monophosphates are well known intermediatesused in several efficient methods for the synthesis of the correspondingNTPs (Burgess and Cook, 2000, Chem. Rev., 100, 2047). As a similarapproach, the 5′ monophosphate of a solid supported, convenientlyprotected oligonucleotide can be used in a similar fashion. 0,O-Bissilyl protected 5′ phosphotriester oligonucleotide can be used for thepreparation of such intermediate. The 2′ ACE strategy seems the mostsuitable for such synthesis, as the 5′ phosphotriester can bedeprotected employing the conditions used for each 5′ step deprotection.The 2′ ACE protected 5′ DMTr 3′ phosphoramidites (103a) and standardsolid supported RNA synthesis conditions can be used for theconstruction of the 5′ OH oligonucleotide (compound 103b), as previouslydiscussed for scheme 5. It is then involved in a phosphitylationreaction using excessO,O-bis-trimethylsilylethyl-N,N-di-isopropylphosphoramidite in solutionin dry acetonitrile. After shaking the solid support with the 0.5 Msolution of phosphitylation reagent, premixed with 0.25 M solution oftetrazole in dry ACN at room temperature for 30 min, the solution iswashed off and the solid support is well rinsed with dry acetonitrile,then further oxidized or sulfurized using standard reagents.Deprotection of the two TSE protecting groups is performed using 1 MTBAF. The resulting 5′ monophosphate oligonucleotide (110c) is firstactivated using either CDI or DPPC, and then a 0.5 M solution oftributylammonium pyrophosphate in DMF is introduced to the solidsupported oligonucleotide and shaken for 24 h, affording thecorresponding triphosphate intermediate (111d). This oligonucleotide TPis then hydrolyzed and deblocked, as previously described for scheme 3,affording the target RNATP or RNA(αS)TP.

Schemes 21 and 22: Another possible path towards RNA(αS)TP andRNA(αS₂)TP involves the use of 5′ H-phosphonothioate orphosphonodithioate oligonucleotides (112c and 114c, schemes 16 and 17,X═O, and X═S, respectively). The latter can be easily prepared afterreacting the 5′ OH solid supported oligonucleotide (compounds b) withdiphenylthiophosphite (120, scheme 16), prepared from commerciallyavailable diphenylphosphite as previously reported (Tashma et al., 1969,J. Org. Chem., 48, 3966). In such manner, 105b (scheme 16) and 103b(scheme 17) are reacted with 0.5 M solution of 120 in dry pyridine for 1h, followed by subsequent hydrolysis (H₂O-NEt₃) or sulfolisys(TMS₂S-NEt₃), affording the corresponding compounds 112c and 114c(schemes 16 and 17, X═O, and X═S, respectively). The latter are furtheroxidized by iodine in the presence of a silylating agent, containingpyridine, (Peterson et al. 2008, Org. Lett., 10, 1703) which allows theformation of the corresponding pyridinium thiophosphoramidate, which isreacted with tributylammonium pyrophosphate in order to providethiotriphosphates (X═O) 113d (scheme 16) and 115d (scheme 17) or thecorresponding dithiotriphosphates (X═S). After deprotection and cleavagefrom solid support using the conditions previously described for schemes5 and 3, the target RNA(αS)TP and RNA(αS₂)TP are obtained.

Synthesis of RNA 5′-Triphosphate

To RNA support (CPG, 200 mg) in a peptide synthesis vessel was addedpyridine (0.5 ml) followed by a solution of2-chloro-1,3,2-benzodioxaphosphorin-4-one (40 mg) in anhydrous1,4-dioxane (2 ml) under an argon atmosphere. The reaction vessel wasplaced on an analog shaker for 5 h. Then excess reagent was removed byapplying a positive pressure of argon. The support was washed withanhydrous 1,4-dioxane (10 ml) and a solution of tributylammoniumpyrophosphate (450 mg) in dry DMF (2 ml) and tributylamine (0.5 ml) wereadded simultaneously. After agitating the reaction vessel on an analogshaker for 22 h, excess reagent was removed by applying positivepressure of argon. To the support 1% iodine solution in pyridine andwater (98:2) was added. After thirty minutes excess reagent was removedby applying positive pressure of argon. The support was washed with cold1M TEAB buffer followed by acetone. The support was allowed to dry andtransferred the support to a 20 ml vial. The support was suspended in amixture of ethanol (1.8 ml) and ammonium hydroxide (5 ml). Afteragitating at room temperature for 15 h, the solution was filtered andthe support was washed with 12 ml of DMSO. The solution containingRNA-triphosphate was cooled at −20 C for 15 min and a cold solution ofHF/TEA (7 ml) was added. The reaction flask was agitated on an analogshaker at room temperature for 8 h. A small sample of the reactionmixture was diluted with water (4 times) and analyzed by analytical HPLCusing Dionex DNAPac PA-100 column. HPLC analysis of the reaction mixtureclearly showed the formation of RNA-triphosphate.

The reaction mixture was diluted to 100 ml with water and the productwas purified using anion exchange column chromatography. The product waseluted using a gradient of 25 mMTris (pH 8.0) to 1M ammonium chloridecontaining 25 mM Tris (pH 8.0) buffer. Appropriate fractions containingthe product were pooled and desalted using reverse phase columnchromatography.

The identity of the RNA 5′-triphosphate is established by Ion-exchangeHPLC analysis and by LC-MS.

Sequence 5′ to 3′ Calc. MW Found MW 1 HO- 6794.34 6792.88AccGAAGuGuuGuuuGuccTsT (M-H)- 2 PPP-O- 7034.25 7033.13AccGAAGuGuuGuuuGuccTsT (M-H)- The lower case letters refer to 2′-OMenucleotides.

In FIG. 1, and ion-exchange HPLC analysis of purified RNA-Triphosphateis depicted.

In FIG. 2, an LC-MS Analysis of RNA 5′-Triphosphate is depicted. Thefollowing data is relevant to the FIG. 2 graphs:

Molecular Absolute Relative Component Weight Abundance Abundance A7033.13 9434 100.00 B 1747.61 4119  43.66 C 2357.51 2393  25.37 D1767.71 1707  18.09 E 2358.26 1398  14.82

Synthesis of 2′-OMe RNA Triphosphate

2′O-Methyl RNA Triphosphate was prepared in a similar manner asdescribed for the synthesis of RNA-triphosphate. This does not requireHF/TEA treatment since it does not contain any silyl groups on the2′-hydroxyl functions.

Synthesis of RNA Thiotriphosphate

RNA-thiotriphosphates are prepared in a similar manner as describedabove. However, iodine solution is substituted with a solution ofphenylacetyl disulfide in 2,6-lutidine.

Methods for Identifying iRNA Agents with Ability to Inhibit or Stimulatethe Immune System.

Modulation of the immune system can be measured for example by (i)measurement of either the mRNA or protein expression levels of acomponent (e.g., a growth factor, cytokine, or interleukin) of theimmune system, e.g., in a cell or in an animal, (ii) measurement of themRNA or protein levels of a protein factor activated by a component ofthe immune system (for example, NFKB), e.g., in a cell or in an animal,(iii) measurement of cell proliferation, e.g., in a tissue explant or atissue of an animal.

Evaluation of the iRNA agent can include incubating the modified strand(with or without its complement, but preferably annealed to itscomplement) with a biological system, e.g., a sample (e.g, a cellculture). The biological sample can be capable of expressing a componentof the immune system. This allows identification of an iRNA agent thathas an effect on the component. In one embodiment, the step ofevaluating whether the iRNA agent modulates, e.g, stimulates orinhibits, an immune response includes evaluating expression of one ormore growth factors, such as a cytokine or interleukin, or cell surfacereceptor protein, in a cell free, cell-based, or animal assay. Proteinlevels can be assayed, for example, by Western blot techniques, flowcytometry, or reporter gene expression (e.g., expression of afluorescent reporter protein, such as green fluorescent protein (GFP)).The levels of mRNA of the protein of interest can be measured byNorthern blot techniques, RNAse Protection Assays, or QualityControl-PCR (QC-PCR) (including quantitative reverse transcriptioncoupled PCR(RT-PCR)) and analogous methods known in the art. RNA and/orprotein levels resulting from target gene expression can be measured atregular time intervals following introduction of the test iRNA agent,and the levels are compared to those following introduction of a controliRNA agent into cells.

In one embodiment, the step of testing whether the modified iRNA agentmodulates, e.g., stimulates, an immune response includes assaying for aninteraction between the iRNA agent and a protein component of the immunesystem, e.g., a growth factor, such as a cytokine or interleukin, or acell surface receptor protein. Exemplary assay methods includecoimmunoprecipitation assays, bead-based co-isolation methods, nucleicacid footprint assays and colocalization experiments such as thosefacilitated by immunocytochemistry techniques.

Cell proliferaton can be monitored by following the uptake of[³H]thymidine or of a fluorescent dye. Cells were plated in a 96-welltissue culture plate and then incubated with the iRNA agent. Forradiometric analysis, [³H]thymidine is added and incubation iscontinued. The cells are subsequently processed on a multichannelautomated cell harvester (Cambridge Technology, Cambridge, Mass.) andcounted in a liquid scintillation beta counter (Beckman Coulter). Forfluorescence-based analysis, a commercially available assay, like theLIVE/DEAD Viability/Cytotoxicity assay from Molecular Probes can beused. The kit identifies live versus dead cells on the basis of membraneintegrity and esterase activity. This kit can be used in microscopy,flow cytometry or microplate assays.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention. Otherembodiments are in the claims.

1-31. (canceled)
 32. An oligonucleotide of formula I, orpharmaceutically acceptable salts or prodrugs thereof:

wherein: Q₂ and Q₃ are each, independently NH, O or S; X and Y are each,independently, OH, O⁻, OR₁, O⁻, SH, S⁻, Se, BH₃, BH₃ ⁻, H, N(R²)₂,alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted; R¹ is independently alkyl, cycloalkyl, aralkyl,aryl, or heteroaryl, each of which may be optionally substituted; R² isindependently hydrogen, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl,each of which may be optionally substituted; Q₄ and Q₅ are eachindependently O, CH₂, CH(Me), C(Me)₂, CHF, CF₂, NH, NR¹, or S; Q₁ is OH,O⁻, OR₁, S⁻, SH, or SR¹; n is 0, 1, 2, 3, 4 or 5; wherein each repeatingunit can be the same or different; A is absent or selected from thegroup consisting of single-stranded oligonucleotide and double-strandedoligonucleotide, each of which may be chemically modified; B is absentor a linker/spacer; and E is a single-stranded oligonucleotide or adouble-stranded oligonucleotide, each of which may be chemicallymodified and/or conjugated with a ligand; with the proviso that when Aand B are both absent and n is 0, 1 or 3, then Q₁, Q₂, Q₃, Q₄, Q₅, X andY cannot all be oxygen.
 33. The oligonucleotide of claim 32, representedby formula (II) or a pharmaceutically acceptable salt or prodrugthereof:

Q₂, Q₃ and Q₃₀ are each, independently NH, O or S; X and Y and Y₁₀ areeach, independently, OH, O⁻, OR¹, O⁻, SH, S⁻, Se, BH₃, BH₃ ⁻, H, N(R²)₂,alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted; R¹ is independently alkyl, cycloalkyl, aralkyl,aryl, or heteroaryl, each of which may be optionally substituted; R² isindependently hydrogen, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl,each of which may be optionally substituted; Q₄ and Q₅ are eachindependently O, CH₂, CH(Me), C(Me)₂, CHF, CF₂, NH, NR₁, or S; Q, is OH,O⁻, OR¹, S⁻, SH, or SR¹; and W is H, OH or -G-L; where G is selectedfrom the group consisting of —CONH—, —NHCO—, —S—S—, —OC(O)NH—,—NHC(O)O—, —NHC(O)NH—, acetal, ketal, —O—N═C—, —NH—N═C—, —S—, —O—,pyrrolidine, morpholine, piperazine and thiazolidine; and where L is aligand; provided that when W is OH, then Q₁, Q₂, Q₃, Q₄, Q₅, Q₃₀, X, Yand Y₁₀ cannot all be oxygen.
 34. The oligonucleotide of claim 32,represented by formula (III) or a pharmaceutically acceptable salt orprodrug thereof:

Q₂ and Q₃ are each, independently NH, O or S; X and Y and Y₁₀ are each,independently, OH, O⁻, OR¹, O⁻, SH, S⁻, Se, BH₃, BH₃ ⁻, H, N(R²)₂,alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted; R¹ is independently alkyl, cycloalkyl, aralkyl,aryl, or heteroaryl, each of which may be optionally substituted; R² isindependently hydrogen, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl,each of which may be optionally substituted; Q₄ and Q₅ are eachindependently O, CH₂, CH(Me), C(Me)₂, CHF, CF₂, NH, NR₁, or S; Q₁ is OH,O⁻, OR¹, S⁻, SH, or SR¹; and W is each independently H, OH or -G-L;where G is selected from the group consisting of —CONH—, —NHCO—, —S—S—,—OC(O)NH—, —NHC(O)O—, —NHC(O)NH—, acetal, ketal, —O—N═C—, —NH—N═C—, —S—,—O—, pyrrolidine, morpholine, piperazine and thiazolidine; and where Lis a ligand.
 35. The oligonucleotide of claim 32, represented by formula(IV) or a pharmaceutically acceptable salt or prodrug thereof:

Q₂, Q₃ and Q₃₀ are each, independently NH, O or S; X and Y and Y₁₀ areeach, independently, OH, O⁻, OR₁, O⁻, SH, S⁻, Se, BH₃, BH₃ ⁻, H, N(R²)₂,alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted; R¹ is independently alkyl, cycloalkyl, aralkyl,aryl, or heteroaryl, each of which may be optionally substituted; R² isindependently hydrogen, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl,each of which may be optionally substituted; Q₄ and Q₅ are eachindependently O, CH₂, CH(Me), C(Me)₂, CHF, CF₂, NR¹, or S; Q₁ is OH, O⁻,OR₁, S⁻, SH, or SR¹; R is H, phosphate or phosphorothioate; W and W₁ areeach independently H, OH, phosphate, phosphorothioate or -G-L; where Gis selected from the group consisting of —CONH—, —NHCO—, —S—S—,—OC(O)NH—, —NHC(O)O—, —NHC(O)NH—, acetal, ketal, —O—N═C—, —NH—N═C—, —S—,—O—, pyrrolidine, morpholine, piperazine and thiazolidine; and where Lis a ligand; provided that when W is OH, then Q₁, Q₂, Q₃, Q₄, Q₅, Q₃₀,X, Y and Y₁₀ cannot all be oxygen.
 36. The oligonucleotide of claim 32,represented by formula (V) or a pharmaceutically acceptable salt orprodrug thereof:

Q₂ and Q₃ are each, independently NH, O or S; X and Y and Y₁₀ are each,independently, OH, O⁻, OR₁, O⁻, SH, S⁻, Se, BH₃, BH₃ ⁻, H, N(R²)₂,alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted; R¹ is independently alkyl, cycloalkyl, aralkyl,aryl, or heteroaryl, each of which may be optionally substituted; R² isindependently hydrogen, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl,each of which may be optionally substituted; Q₄ and Q₅ are eachindependently O, CH₂, CH(Me), C(Me)₂, CHF, CF₂, NR¹, or S; Q₁ is OH, O⁻,OR₁, S⁻, SH, or SR¹; R is H, phosphate or phosphorothioate; W and W₁ areeach independently H, OH, phosphate, phosphorothioate or -G-L; where Gis selected from the group consisting of —CONH—, —NHCO—, —S—S—,—OC(O)NH—, —NHC(O)—, —NHC(O)NH—, acetal, ketal, —O—N═C—, —NH—N═C—, —S—,—O—, pyrrolidine, morpholine, piperazine and thiazolidine; and where Lis a ligand.
 37. The oligonucleotide of claim 32, represented by formula(VI) or a pharmaceutically acceptable salt or prodrug thereof:

Q₂, Q₃ and Q₃₀ are each, independently NH, O or S; X and Y and Y₁₀ areeach, independently, OH, O⁻, OR₁, O⁻, SH, S⁻, Se, BH₃, BH₃ ⁻, H, N(R²)₂,alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted; R¹ is independently alkyl, cycloalkyl, aralkyl,aryl, or heteroaryl, each of which may be optionally substituted; R² isindependently hydrogen, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl,each of which may be optionally substituted; Q₄ and Q₅ are eachindependently O, CH₂, CH(Me), C(Me)₂, CHF, CF₂, NR¹, or S; Q₁ is OH, O⁻,OR₁, S⁻, SH, or SR¹; W, W₁ and W₂ are each independently H, OH,phosphate, phosphorothioate or -G-L; where G is selected from the groupconsisting of —CONH—, —NHCO—, —S—S—, —OC(O)NH—, —NHC(O)O—, —NHC(O)NH—,acetal, ketal, —O—N═C—, —NH—N═C—, —S—, —O—, pyrrolidine, morpholine,piperazine and thiazolidine; and where L is a ligand; and Linker/spaceris selected from the group consisting of phosphate, phosphorothioate,phosphorodithioate, alkylphosphonate, amide, ester, disulfide,thioether, oxime, hydrazone, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, and heteroaryl.
 38. The oligonucleotide of claim 32,represented by formula (VII) or a pharmaceutically acceptable salt orprodrug thereof:

Q₂ and Q₃ are each, independently NH, O or S; X and Y and Y₁₀ are each,independently, OH, O⁻, OR¹, O⁻, SH, S⁻, Se, BH₃, BH₃ ⁻, H, N(R²)₂,alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may beoptionally substituted; R¹ is independently alkyl, cycloalkyl, aralkyl,aryl, or heteroaryl, each of which may be optionally substituted; R² isindependently hydrogen, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl,each of which may be optionally substituted; Q₄ and Q₅ are eachindependently O, CH₂, CH(Me), C(Me)₂, CHF, CF₂, NR¹, or S; Q₁ is OH, O⁻,OR¹, S⁻, SH, or SR¹; W, W, and W₂ are each independently H, OH,phosphate, phosphorothioate or -G-L; where G is selected from the groupconsisting of —CONH—, —NHCO—, —S—S—, —OC(O)NH—, —NHC(O)O—, —NHC(O)NH—,acetal, ketal, —O—N═C—, —NH—N═C—, —S—, —O—, pyrrolidine, morpholine,piperazine and thiazolidine; and where L is a ligand; and Linker/spaceris selected from the group consisting of phosphate, phosphorothioate,phosphorodithioate, alkylphosphonate, amide, ester, disulfide,thioether, oxime, hydrazone, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, and heteroaryl.
 39. The oligonucleotide of claim 32,wherein the single- or double-stranded oligonucleotide comprises atleast one modified nucleotide.
 40. The oligonucleotide of claim 39,wherein at least one of said modified nucleotides is selected from thegroup consisting of a 2′-O-methyl modified nucleotide, a nucleotidecomprising a 5′-phosphorothioate group, and a terminal nucleotide linkedto a cholesteryl derivative or dodecanoic acid bisdecylamide group. 41.The oligonucleotide of claim 39, wherein at least one of said modifiednucleotides is selected from the group of a 2′-fluoro nucleotide, a2′-O-alkyl nucleotide, a 2′-O-alkoxyalkyl nucleotide, a 2′-O-allylnucleotide, a 2′ 0-propyl nucleotide, a 2′-O-methylated nucleotide(2′-O-Me), a 2′-deoxy nucleotide, a 2′-deoxyfluoro nucleotide, a2′-O-methoxyethyl nucleotide (2′-O-MOE), a 2′-O—N-MeAcetamide nucleotide(2′-O-NMA), a 2′-β-dimethylaminoethyloxyethyl nucleotide (2′-O— DMAEOE),a 2′-aminopropyl, a 2′-hydroxy, a 2′-ara-fluoro, a 3′-amidate (3′—NH inplace of 3′-0), a locked oligonucleotide (LNA), an extended ethyleneoligonucleotide (ENA), a hexose oligonucleotide (HNA), or a cyclohexeneoligonucleotide (CeNA).
 42. The oligonucleotide of claim 32, wherein theoligonucleotide is comprised in a viral vector.
 43. The oligonucleotideof claim 32, wherein the oligonucleotide binds to RIG-I.
 44. Acomposition comprising the oligonucleotide of claim 32 and at least oneagent selected from the group consisting of an immunostimulatory agent,an anti-viral agent, an anti-bacterial agent, an anti-tumor agent, agene-silencing agent, an anti-tumor therapy, and combinations thereof.45. The composition of claim 44, wherein the agent is retinoic acid,type I IFN, or a combination thereof.
 46. A method of using theoligonucleotide of claim 32 for inducing apoptosis of tumor cells,inducing an anti-viral response, inducing an anti-bacterial response,and/or inducing an anti-tumor response in a vertebrate animal.
 47. Themethod of claim 46, wherein the anti-viral response, the anti-bacterialresponse and/or the anti-tumor response comprise type I IFN production,IL-18 production, and/or IL-1β production.
 48. A composition comprisingthe oligonucleotide of claim 32 and at least one antigen for inducing animmune response against an antigen in a vertebrate animal.
 49. Thecomposition of claim 48, wherein the oligonucleotide is covalentlylinked to the at least one antigen.
 50. A method of using theoligonucleotide of claim 32 for the preparation of a medicament forpreventing and/or treating a disease and/or disorder selected from thegroup consisting of viral infection, bacterial infection, parasiticinfection, tumor, multiple sclerosis, allergy, autoimmune diseases,immunosuppression, and immunodeficiency in a vertebrate animal.
 51. Amethod of using the oligonucleotide of claim 32 for the preparation of amedicament for inducing apoptosis of tumor cells, inducing an anti-viralresponse, inducing an anti-bacterial response, and/or inducing ananti-tumor response in a vertebrate animal.
 52. The method of claim 51,wherein the anti-viral response, the anti-bacterial response, and/or theanti-tumor response comprise type I IFN production, IL-18 production,and/or IL-1β production.
 53. A pharmaceutical composition comprising theoligonucleotide of claim 32 and a pharmaceutically acceptable carrier.54. A pharmaceutical package, comprising the pharmaceutical compositionof claim 53 and an instruction for use.
 55. A combined preparation,comprising the oligonucleotide of claim 32 and at least one agentselected from the group consisting of an immunostimulatory agent, ananti-viral agent, an anti-bacterial agent, an anti-tumor agent, and agene silencing agent, wherein the oligonucleotide and the at least oneagent are administered simultaneously, separately, or sequentially. 56.The combined preparation of claim 55, wherein the agent is retinoicacid, type I IFN, or a combination thereof.
 57. A pharmaceuticalpackage, comprising the combined preparation of claim 55 and aninstruction for use.
 58. A method of using the oligonucleotide of claim32 for the preparation of a bacterial RNA for preventing and/or treatinga disease and/or disorder selected from the group consisting of viralinfection, bacterial infection, parasitic infection, tumor, multiplesclerosis, allergy, autoimmune diseases, immunosuppression, andimmunodeficiency in a vertebrate animal.
 59. A process of preparing anoligonucleotide molecule having one or more ribonucleotides that containa triphosphosphate or a triphosphate analog, comprising the steps of:(a) protecting the 2′ hydroxyl moiety of one or more ribonucleotideswith a fluoride labile group or a fluoride non-labile group; (b)converting the desired terminal hydroxyl moiety to a triphosphate ortriphosphate analog with a reagent selected from the group consistingof:

wherein: R₁₀₀ is independently electron withdrawing group (EWG); R₂₀₀and R₃₀₀ are each independently haloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocycle, substituted heterocyclo; Z₁₀ is O, S, Se, BH₃ or NR; X₄₀ isCl, dialkylamine or cyclic amine; X₁₀ is Cl, O-aryl or O-substitutedaryl; Y₁₀ and Y₂₀ are independently O-substituted alkyl; dialkylamine orcyclic amine, wherein the nitrogen is connected to the phosphorus; X₂₀and X₃₀ is independently O, CH₂, S, NR′, wherein R′ is H or aliphatic; nis 1, 2, 3, 4, or 5; and s is 0, 1, 2 or 3; (c) synthesizing saidoligonucleotide molecule using a method selected from the groupconsisting of solid phase phosphoramidite, solution phasephosphoramidite, solid phase H-phosphonate, solution phaseH-phosphonate, hybrid phase phosphoramidite, and hybrid phaseH-phosphonate-based synthetic methods; and (d) removing the protectinggroup(s) and/or solid support.
 60. The pharmaceutical composition ofclaim 53, adapted for delivery by a mode selected from the groupconsisting of intraocular injection, oral ingestion, enteralapplication, inhalation, topical application, subcutaneous injection,intramuscular injection, intraperitoneal injection, intrathecalinjection, intratrachael injection, and intravenous injection.
 61. A kitcomprising a oligonucleotide of claim 32 in a labeled package, whereinthe label on said package indicates that said oligonucleotide can beused against at least one virus.
 62. The kit of claim 61, wherein saidkit is approved by a regulatory agency for use in humans.
 63. An assayfor identifying an anti-viral or an antibacterial response, comprising atest sample and a test agent, wherein the test agent comprises anoligonucleotide of claim 32.